Bioorthogonal Turn-on Probes

ABSTRACT

or a salt thereof, wherein: F is a fluorophore, L is a conjugated linker, and Tz is a substituted or unsubstituted tetrazine; wherein the linker bridges the Tz and F moieties in a single conjugated pi-system. Also provided herein are methods of using the compounds provided herein for biomedical imaging.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/889,647, filed Nov. 6, 2015, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2014/036977, filed on May 6, 2014, which claims priority toU.S. Application Ser. No. 61/819,913, filed on May 6, 2013, both ofwhich are incorporated by reference in their entirety.

TECHNICAL FIELD

This present application relates to fluorescent tetrazine-containingcompounds consisting of a single pi-system. Also provided herein aremethods of using the compounds provided herein for biomedical imaging.

BACKGROUND

Visualizing biomolecular processes has been enhanced by combiningfluorophores with bioorthogonal chemistry, resulting in new tools tostudy the complex biochemical milieu of living cells and organisms. (N.K. Devaraj, et al., Angew. Chem. 2009, 121, 7147-7150; M. Boyce and C.R. Bertozzi, Nat. Methods 2011, 8, 638; and C. L. Droumaguet and C.Wang, Q. Wang, Chem. Soc. Rev. 2010, 39, 1233.) The resulting probeshave been applied to image glycosylation and phospholipid uptake,cellular proteins, and intracellular drug distribution. (P. Shieh, etal., J. Am. Chem. Soc. 2012, 134, 17428; S. T. Laughlin, et al., Science2008, 320, 664; M. J. Hangauer and C. R. Bertozzi, Angew. Chem. 2008,120, 2353; J. Yang, et al, Angew. Chem. 2012, 124, 7594-7597; K. Lang,et al., Nat. Chem. 2012, 4, 298; D. S. Liu, et al., J. Am. Chem. Soc.2012, 134, 792; N. K. Devaraj, et al., Bioconjug. Chem. 2008, 19, 2297;J. Z. Yao, et al., J. Am. Chem. Soc. 2012, 134, 3720; K. S. Yang, etal., Angew. Chem. 2012, 124, 6702-6707; G. Budin, et al., Angew. Chem.2011, 123, 9550-9553; T. Reiner, et al, Chembiochem 2010, 11, 2374.)

Bioorthogonal “click” chemistries are widely used in chemical biologyfor a myriad of applications such as activity based protein profiling,crosslinking of proteins, monitoring cell proliferation, generation ofnovel enzyme inhibitors, monitoring the synthesis of newly formedproteins, protein target identification, and studying glycan processing.Perhaps the most fascinating applications involve using thesebioorthogonal chemistries to assemble molecules in the presence ofliving systems such as live cells or even whole organisms (Baskin etal., 2007, Proc Natl Acad Sci USA, 104, 16793-7; Laughlin et al., 2008,Science, 320, 664-7; Prescher and Bertozzi, 2005, Nat Chem Biol, 1,13-21; Neef and Schultz, 2009, Angew Chem Int Ed Engl, 48, 1498-500;Ning et al., 2008, Angewandte Chemie-International Edition, 47,2253-2255). These latter applications require that the chemistry benon-toxic and possess kinetics that allow fast reaction to occur withmicromolar concentrations of reagents in a time span of minutes tohours.

To fulfill these criteria, various “copper-free” click chemistries havebeen reported, such as the strain-promoted azide-alkyne cycloadditionand the Staudinger ligation, to react with azides on the surface of livecells both in culture and in in vivo systems such as mice and zebrafish(Prescher and Bertozzi, 2005, Nat Chem Biol, 1, 13-21). However, todate, the application of “click” chemistry in living systems, has beenlargely limited to extracellular targets and no technique has shownreliable ability to specifically label and image intracellular targets(Baskin and Bertozzi, 2007, QSAR Comb. Sci., 26, 1211-1219). The reasonsfor this are likely several. In addition to fulfilling the stability,toxicity, and chemoselectivity requirements of “click” chemistry,intracellular live cell labeling requires reagents that can easily passthrough biological membranes and kinetics that enable rapid labelingeven with the low concentrations of agent that make it across the cellmembrane. Additionally, a practical intracellular bioorthogonal couplingscheme would need to incorporate a mechanism by which the fluorescenttag increases in fluorescence upon covalent reaction to avoidvisualizing accumulated but unreacted imaging probes (i.e. background).This “turn-on” would significantly increase the signal-to-backgroundratio, which is particularly relevant to imaging targets inside livingcells since a stringent washout of unreacted probe is not possible.

In previous years a number of elegant probes have been introduced whosefluorescence increases after azide-alkyne cycloaddition or staudingerligation coupling reactions (Sivakumar et al., 2004, Org Lett, 6,4603-6; Zhou and Fahrni, 2004, J Am Chem Soc, 126, 8862-3; Hangauer andBertozzi, 2008, Angew Chem Int Ed Engl, 47, 2394-7; Lemieux et al.,2003, J Am Chem Soc, 125, 4708-9). Most of these strategies eitherrequire a reactive moiety intimately attached to the fluorophore thusrequiring synthesis of new fluorophore scaffolds or take advantage of aFRET based activation requiring appendage of an additional molecule thatcan act as an energy transfer agent. Furthermore, most probes utilizingthese popular coupling schemes have to date been unable to labelintracellular targets in live cells.

The bioorthogonal Diels-Alder reaction is compatible with aqueousenvironments and has second order rate constants that are known to beenhanced up to several hundred-fold in aqueous media in comparison toorganic solvents. (Rideout D C et al., 1980, J Am Chem Soc102:7816-7817; Graziano G, 2004, J Phys Org Chem 17:100-101). ManyDiels-Alder reactions are reversible, therefore, they may not besuitable for biological labeling. (Kwart et al., 1968, Chem Rev68:415-447), however, the inverse electron demand Diels-Aldercycloaddition of olefins with tetrazines results in irreversiblecoupling giving dihydropyridazine products. During this reaction,dinitrogen is released in a retro Diels-Alder step. (Sauer J et al.,1965, Chem Ber 998:1435-1445). A variety of tetrazines and dienophilesincluding cyclic and linear alkenes or alkynes have been studied in thisreaction. Selection of the appropriate reaction partners, allows fortuning of the coupling rate by several orders of magnitude. (Balcar J etal., 1983, Tet Lett 24:1481-1484; Thalhammer F et al., 1990, Tet Lett47:6851-6854). See also US 2006/0269942, WO 2007/144200, and US2008/0181847.

SUMMARY

In some previously described fluorophores, the capacity of in situchemical conjugation has been paired with fluorogenic turn-on, wherebyfluorophore emission increases upon reaction with its bioorthogonalcounterpart (“turn-on” probes). See, e.g., N. K. Devaraj, et al., Angew.Chem. 2009, 121, 7147-7150. This has the very attractive feature ofreducing background fluorescence when doing in vivo imaging, potentiallyallowing real time imaging, without washing or clearance steps. Existingmethods that exploit azide-phosphine, azide-alkyne, or inverse electrondemand Diels-Alder tetrazine cycloadditions have not provided probeswith high fluorescence turn-on ratios and fast kinetics without using acatalyst.

Provided herein are compounds of Formula (I) or (II):

F-L-Tz   (I)

F-Tz   (II)

or a salt thereof,

-   wherein:-   F is a fluorophore;-   L is a conjugated linker; and-   Tz is a substituted or unsubstituted tetrazine;-   wherein the linker bridges the Tz and F moieties in a single    conjugated pi-system.

Compound of Formula (I) or (II) can be reacted with a dienophile toprepare compounds of Formula (III) or (IV):

F-L-Z   (III)

F-Z   (IV)

or a salt thereof,

-   wherein:-   F is a fluorophore;-   L is a conjugated linker; and-   Z is a moiety comprising the reaction product of a diene and a    dienophile, wherein the diene is a tetrazine or a derivative    thereof;-   wherein the linker bridges the Z and F moieties in a single    conjugated pi-system.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a fluorescence emission spectra for compound 4b inacetonitrile at baseline (black) and after addition of TCO (green);excitation at 490 nm.

FIG. 2 illustrates normalized fluorescence turn-on after addition of TCO(240 μM) to a solution of the indicated fluorophore (1 μM) inacetonitrile; initial intensity 1.0 a.u.

FIG. 3 is a photograph showing equimolar solutions of compound 2a (atleft) and 2a plus TCO (at right) under excitation by a handheld UV lamp.

FIG. 4 is a 3D model of compound 4a, illustrating a twisted phenyllinker between the BODIPY and Tz chromophores and the orientation of thedonor and acceptor transition dipoles (gray arrows).

FIG. 5 shows the absorption and fluorescence emission spectra of 4b intoluene and acetonitrile.

FIG. 6 illustrates the fluorogenic activation of 4b in water.

FIG. 7 illustrates the biological application of the compounds providedherein. FIG. 7A shows fluorogenic imaging of EGFR expression on bothfixed and live A431 cells. FIG. 7B are fluorogenic live-cell images ofintracellular nanoparticles internalized by RAW 264.7 cells.

FIG. 8 illustrates a tetrazine transition dipole as collinear with orparallel to the transition dipole of the fluorophore.

FIG. 9 shows the fluorogenic activation of HELIOS probes upon reactionwith TCOc (H=HELIOS).

FIG. 10 shows the fluorescence emission spectra of HELIOS 400Me in PBS(pH 7.4), acetonitrile (dielectric constant 37.5), and toluene(dielectric constant 2.4).

FIG. 11A shows no wash fluorogenic imaging of EGFR expression on A431cells using HELIOS 370H. Bright, membrane specific staining is visiblewithin seconds, peaks within 3 minutes and is stable thereafter. FIG.11B shows cells imaged with HELIOS 370H (left) and control cells (right)exhibiting autofluorescence at baseline, prior to addition of dye.

FIGS. 12A-12B illustrates no-wash fluorogenic imaging of intracellulartargets. FIG. 12A shows mitochondrial imaging of OVCA-429 cells withRFP-tagged mitochondria were incubated with an anti-mitochondria-TCOantibody, rinsed briefly, and then imaged after addition of 100 nmHELIOS-388H in PBS. FIG. 12B shows actin imaging of COS-1 cellsincubated with phalloidin-TCO (1 μg/mL) and DRAQ5 nuclear counterstain(1 μM, BioStatus), rinsed briefly, and then imaged upon addition of theindicated HELIOS probe at 100 nM. Control images were collected atmatched dye concentrations in the absence of phalloidin-TCO.

FIG. 13 illustrates in vivo imaging of a HELIOS probe as describedherein.

DETAILED DESCRIPTION Definitions

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. As used herein, the term “about” ismeant to account for variations due to experimental error. Allmeasurements reported herein are understood to be modified by the term“about”, whether or not the term is explicitly used, unless explicitlystated otherwise. As used herein, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

The term “alkyl” includes straight-chain alkyl groups (e.g., methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.)and branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl,etc.). In certain embodiments, a straight chain or branched chain alkylhas twelve or fewer carbon atoms in its backbone (e.g., C₁-C₁₂ forstraight chain; C₃-C₁₂ for branched chain). The term C₁-C₁₂ includesalkyl groups containing 1 to 12 carbon atoms.

The term “alkenyl” includes aliphatic groups that may or may not besubstituted, as described above for alkyls, containing at least onedouble bond and at least two carbon atoms. For example, the term“alkenyl” includes straight-chain alkenyl groups (e.g., ethenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, anddecenyl) and branched-chain alkenyl groups. In certain embodiments, astraight chain or branched chain alkenyl group has twelve or fewercarbon atoms in its backbone (e.g., C₂-C₁₂ for straight chain; C₃-C₁₂for branched chain). The term C₂-C₁₂ includes alkenyl groups containing2 to 12 carbon atoms.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond and two carbon atoms. Forexample, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, and decynyl) and branched-chain alkynyl groups. In certainembodiments, a straight chain or branched chain alkynyl group has twelveor fewer carbon atoms in its backbone (e.g., C₂-C₁₂ for straight chain;C₃-C₁₂ for branched chain). The term C₂-C₁₂ includes alkynyl groupscontaining 2 to 12 carbon atoms.

The term “alkoxy” is used in its conventional sense, and refers to alkylgroups linked to molecules via an oxygen atom. In some embodiments, analkoxy has twelve or fewer carbon atoms in its backbone (e.g., a C₁-C₁₂alkoxy). For example, C₁-C₁₀, C₁-C₈, C₁-C₆, C₁-C₄, C₁-C₃, or C₁-C₂.Non-limiting examples of an alkoxy group include methoxy, ethoxy,propoxy, butoxy, and hexoxy.

The terms “halo” or “halogen”, used alone or in combination with otherterms, refers to fluoro, chloro, bromo and iodo.

The term “haloalkyl” refers to an alkyl group in which one or more ofthe hydrogen atoms has been replaced by a halogen atom. The term“C_(n)-C_(m) haloalkyl” refers to a C_(n)-C_(m) alkyl group having n tom carbon atoms, and from at least one up to {2(n to m)+1} halogen atoms,which may either be the same or different. In some embodiments, thehalogen atoms are fluoro atoms. In some embodiments, the haloalkyl grouphas 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF₃,C₂F₅, CHF₂, CCl₃, CHCl₂, C₂Cl₅ and the like. In some embodiments, thehaloalkyl group is a fluoroalkyl group.

The term “haloalkoxy”, employed alone or in combination with otherterms, refers to a group of formula —O-haloalkyl, wherein the haloalkylgroup is as defined above. The term “C_(n)-C_(m) haloalkoxy” refers to ahaloalkoxy group, the haloalkyl group of which has n to m carbons.Example haloalkoxy groups include trifluoromethoxy and the like. In someembodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbonatoms.

The term “amino” refers to a group of formula —NH₂.

The term “carbamyl” refers to a group of formula —C(O)NH₂.

The term “carbonyl”, employed alone or in combination with other terms,refers to a —C(═)— group, which also may be written as C(O).

The term “oxo” refers to oxygen as a divalent substituent, forming acarbonyl group, or attached to a heteroatom forming a sulfoxide orsulfone group, or an N-oxide group.

The term “aromatic” refers to a carbocycle or heterocycle having one ormore polyunsaturated rings having aromatic character (i.e., having(4n+2) delocalized π (pi) electrons where n is an integer).

The term “carbocyclyl” includes a cyclic aliphatic group which may besaturated or unsaturated. For example, carbocyclyl groups includecyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Insome embodiments, carbocyclyls have from 3-8 carbon atoms in their ringstructure, for example, they can have 3, 4, 5 or 6 carbons in the ringstructure.

In general, the term “aryl” includes groups, including 5- and 6-memberedsingle-ring aromatic groups, such as benzene and phenyl. Furthermore,the term “aryl” includes multicyclic aryl groups, e.g., tricyclic,bicyclic, such as naphthalene and anthracene.

The term “heteroaryl” includes groups, including 5- and 6-memberedsingle-ring aromatic groups, that have from one to four heteroatoms, forexample, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole,triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like. Furthermore, the term“heteroaryl” includes multicyclic heteroaryl groups, e.g., tricyclic,bicyclic, such as benzoxazole, benzodioxazole, benzothiazole,benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,isoquinoline, napthyridine, indole, benzofuran, purine, benzofuran,quinazoline, deazapurine, indazole, or indolizine.

The term “heterocyclyl” includes non-aromatic groups, including but notlimited to, 3- to 10-membered single or multiple non-aromatic ringshaving one to five heteroatoms, for example, oxetane, piperazine,pyrrolidine, piperidine, or homopiperazine.

The term “substituted” means that an atom or group of atoms replaceshydrogen as a “substituent” attached to another group. For aryl andheteroaryl groups, the term “substituted”, unless otherwise indicated,refers to any level of substitution, namely mono, di, tri, tetra, orpenta substitution, where such substitution is permitted. Thesubstituents are independently selected, and substitution may be at anychemically accessible position. In some cases, two sites of substitutionmay come together to form a 3-10 membered carbocyclyl or heterocyclylring.

Substituents include, but are not limited to, Cy¹, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, halo, C₁₋₆ haloalkyl, CN, NO₂, OR^(a1), SR^(a1),C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), C(═NR^(e1))NR^(c1)R^(d1),NR^(c1)C(═NR^(e1))NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), NR^(c1)C(O)NR^(c1)R^(d1), NR^(c1)S(O)R^(b1),NR^(c1)S(O)₂R^(b1), NR^(c1)S(O)₂NR^(c1)R^(d1), S(O)R^(b1),S(O)NR^(c1)R^(d1), S(O)₂R^(b1) and S(O)₂NR^(c1)R^(d1).

each Cy¹ is independently C₆₋₁₀ aryl, C₃₋₁₀ carbocyclyl, 5-10 memberedheteroaryl or 4-10 membered heterocyclyl, each of which is unsubstitutedor substituted by 1, 2, 3, 4 or 5 substituents independently selectedfrom C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), C(═NR^(c1))NR^(c1)R^(d1),NR^(c1)C(═NR^(e1))NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(d1),NR^(c1)C(O)OR^(a1), NR^(c1)C(O)NR^(c1)R^(d1), NR^(c1)S(O)R^(b1),NR^(c1)S(O)₂R^(b1), NR^(c1)S(O)₂NR^(c1)R^(d1), S(O)R^(b1),S(O)NR^(c1)R^(d1), S(O)₂R^(b1), S(O)₂NR^(c1)R^(d1) and oxo;

each R^(a1), R^(b1), R^(c1), R^(d1), is independently selected from H,C₁₋₆ alkyl, C₁₋₄ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₀ aryl,C₃₋₁₀ carbocyclyl, 5-10 membered heteroaryl, 4-10 membered heterocyclyl,C₆₋₁₀ aryl-C₁₋₄ alkyl, C₃₋₁₀ carbocyclyl-C₁₋₄ alkyl, (5-10 memberedheteroaryl)-C₁₋₄ alkyl or (4-10 membered heterocyclyl)-C₁₋₄ alkyl,wherein said C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₀ aryl, C₃₋₁₀carbocyclyl, 5-10 membered heteroaryl, 4-10 membered heterocyclyl, C₆₋₁₀aryl-C₁₋₄ alkyl, C₃₋₁₀ carbocyclyl-C₁₋₄ alkyl, (5-10 memberedheteroaryl)-C₁₋₄ alkyl and (4-10 membered heterocyclyl)-C₁₋₄ alkyl isoptionally substituted with 1, 2, 3, 4, or 5 substituents independentlyselected from C₁₋₄ alkyl, C₁₋₄ haloalkyl, halo, CN, OR^(a4), SR^(a4),C(O)R^(b4), C(O)NR^(c4)R^(d4), C(O)OR^(a4), OC(O)R^(b4),OC(O)NR^(c4)R^(d4), NR^(c4)R^(d4), NR^(c4)C(O)R^(b4),NR^(c4)C(O)NR^(c4)R^(d4), NR^(c4)C(O)OR^(a4), C(═NR^(e4))NR^(c4)R^(d4),NR^(c4)C(═NR^(e4))NR^(c4)R^(d4), S(O)R^(b4), S(O)NR^(c4)R^(d4),S(O)₂R^(b4), NR^(c4)S(O)₂R^(b4), NR^(c4)S(O)₂NR^(c4)R^(d4) andS(O)₂NR^(c4)R^(d4);

each R^(a4), R^(b4), R^(c4) and R^(d4) is independently selected from H,C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₂₋₄ alkenyl and C₂₋₄ alkynyl, wherein saidC₁₋₄ alkyl, C₂₋₄ alkenyl, and C₂₋₄ alkynyl, is optionally substitutedwith 1, 2, or 3 substituents independently selected from OH, CN, amino,halo, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylthio, C₁₋₄ alkylamino, di(C₁₋₄alkyl)amino, C₁₋₄ haloalkyl, and C₁₋₄ haloalkoxy;

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, —CH₂O— is equivalent to—OCH₂—. In some embodiments, one or more substituents can be a groupreactive with a biologically active molecule or a detectable agent.

The abbreviation “PEG” as used herein refers to a polyethylene glycolpolymer.

A “reactive moiety” as used herein refers to a reactive moiety forconjugation between the fluorophore and/or the dienophile and abiologically active compound (e.g., an antibody). Non-limiting examplesof reactive moietys include hydroxyl, amine, thiol, carboxyl, aldehyde,glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinylsulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide,

wherein n is an integer from 1 to 10, and

As used herein, chemical structures which contain one or morestereocenters depicted with dashed and bold bonds (i.e.,

) are meant to indicate absolute stereochemistry of the stereocenter(s)present in the chemical structure. As used herein, bonds symbolized by asimple line do not indicate a stereo-preference. Unless otherwiseindicated, chemical structures, which include one or more stereocenters,illustrated herein without indicating absolute or relativestereochemistry encompass all possible stereoisomeric forms of thecompound (e.g., diastereomers, enantiomers) and mixtures thereof (e.g.,racemic mixtures). Structures with a single bold or dashed line, and atleast one additional simple line, encompass a single enantiomeric seriesof all possible diastereomers.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An exemplary method includesfractional recrystallization using a chiral resolving acid which is anoptically active, salt-forming organic acid. Suitable resolving agentsfor fractional recrystallization methods are, for example, opticallyactive acids, such as the D and L forms of tartaric acid,diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malicacid, lactic acid, or the various optically active camphorsulfonic acidssuch as camphorsulfonic acid. Other resolving agents suitable forfractional crystallization methods include stereoisomerically pure formsof methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent compositions canbe determined by one skilled in the art.

The term “counter anion” as used herein is intended to include anycounter anions of inorganic and organic acids. Exemplary anions include,but are not limited to: chloride, bromide, iodide, nitrate, sulfate,bisulfate, sulfite, bisulfite, phosphate, acid phosphate, perchlorate,chlorate, chlorite, hypochlorite, periodate, iodate, iodite, hypoiodite,carbonate, bicarbonate, isonicotinate, acetate, trichloroacetate,trifluoroacetate, lactate, salicylate, citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, trifluoromethanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, p-trifluoromethylbenzenesulfonate,hydroxide, aluminates, and borates.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “salt” includes any ionic form of a compound and one or morecounter-ionic species (cations and/or anions). Salts also includezwitterionic compounds (i.e., a molecule containing one more cationicand anionic species, e.g., zwitterionic amino acids). Counter ionspresent in a salt can include any cationic, anionic, or zwitterionicspecies. Exemplary anions include, but are not limited to: chloride,bromide, iodide, nitrate, sulfate, bisulfate, sulfite, bisulfite,phosphate, acid phosphate, perchlorate, chlorate, chlorite,hypochlorite, periodate, iodate, iodite, hypoiodite, carbonate,bicarbonate, isonicotinate, acetate, trichloroacetate, trifluoroacetate,lactate, salicylate, citrate, tartrate, pantothenate, bitartrate,ascorbate, succinate, maleate, gentisinate, fumarate, gluconate,glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate,trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, p-trifluoromethylbenzenesulfonate, hydroxide,aluminates, and borates.

Exemplary cations include, but are not limited to: monovalent alkalimetal cations, such as lithium, sodium, potassium, and cesium, anddivalent alkaline earth metals, such as beryllium, magnesium, calcium,strontium, and barium. Also included are transition metal cations, suchas gold, silver, copper and zinc, as well as non-metal cations, such asammonium salts.

Also provided herein are pharmaceutically acceptable salts of thecompounds described herein. As used herein, “pharmaceutically acceptablesalts” refers to derivatives of the disclosed compounds wherein theparent compound is modified by converting an existing acid or basemoiety to its salt. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. The pharmaceutically acceptable saltsof the compounds provided herein include the conventional non-toxicsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. The pharmaceutically acceptable salts of thecompounds provided herein can be synthesized from the parent compoundwhich contains a basic or acidic moiety by conventional chemicalmethods. Generally, such salts can be prepared by reacting the free acidor base forms of these compounds with a stoichiometric amount of theappropriate base or acid in water or in an organic solvent, or in amixture of the two; in some embodiments, a non-aqueous media like ether,ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, orbutanol) or acetonitrile (ACN) can be used. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., 1985, p. 1418 and Journal of PharmaceuticalScience, 66, 2 (1977). Conventional methods for preparing salts aredescribed, for example, in Handbook of Pharmaceutical Salts: Properties,Selection, and Use, Wiley-VCH, 2002.

The term “essentially pure” refers to chemical purity of a compoundprovided herein that may be substantially or essentially free of othercomponents which normally accompany or interact with the compound priorto purification. By way of example only, a compound may be “essentiallypure” when the preparation of the compound contains less than about 30%,less than about 25%, less than about 20%, less than about 15%, less thanabout 10%, less than about 5%, less than about 4%, less than about 3%,less than about 2%, or less than about 1% (by dry weight) ofcontaminating components. Thus, an “essentially pure” compound may havea purity level of about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99% or greater. Forthe purposes of this document, preparations of functionalized polymersor conjugates differing only in the length of their polymer chain areconsidered to be essentially pure. An essentially pure compound may beobtained using chromatographic purification methods.

As used herein, a “turn-on ratio” is a measure of the increase influorescence associated with probe activation and is the ratio of thefluorescence after and before activation of a compound provided herein.For example, a turn-on ratio for a compound of Formula (III) can becalculated by taking the ratio of the fluorescence intensity of acompound of Formula (III) over the fluorescence intensity of thecorresponding compound of Formula (I). See, e.g., Thompson, M. A. etal., Methods Enzymol 2010 475: 27-59.

As used herein, “conjugated” refers to a compound having alternatingsingle and multiple bonds and containing a system of connectedp-orbitals with delocalized electrons.

Compounds

A fluorogenic probe enables the intrinsic fluorescence of a chromophoreto be stably, but reversibly, suppressed. This can be accomplished bymaking customized modifications that directly perturb the intrinsicfluorophore, or, more generally, by linking a fluorophore to moietiesthat can quench its fluorescence via photoinduced electron transfer(PET) or via energy transfer, e.g. Förster resonance energy transfer(FRET). Subsequent disruption of the quenching group restores the lightemission. An optimal probe of this type thus requires both highlyefficient quenching and a facile, selective mechanism to modulate thequencher—either by cleavage of the linker or chemical reaction of thequencher itself.

Tetrazine-based probes achieve their fluorogenic turn-on by the latterroute, as the tetrazine (Tz) chromophore can be characterized as both aquencher (via energy transfer) and a bioorthogonal reactant. Firstgeneration Tz-fluorophore probes relied on FRET, a through-spacetransfer mechanism that depends on distance, molecular alignment, aswell as the intensity and alignment of emission-absorption bands, all ofwhich can limit energy transfer efficiency. As a result, although theypossess many desirable characteristics, including very fast, catalystfree, chemical reactivity and high brightness after turn-on, theseprobes were limited by fluorescence turn-on ratios of just 10-20 fold(i.e. 90-95% baseline quenching). Similar tradeoffs limit most PET-basedprobes in the literature, as many highly efficient quenchers have nointrinsic bioorthogonal reactivity, and reactive moietys, such asazide-modified fluorophores, have modest quenching ability andcomparatively slow reaction kinetics.

In contrast, through-bond energy transfer (TBET) enables the Tz-basedprobes provided herein to display both exceptional turn-on and chemicalreactivity. In the present system, TBET is markedly more efficient thanFRET, achieving turn-on ratios of greater than 10² and in some casesgreater than 10³ (>99.9% baseline quenching). Several practical featurescan enhance TBET relative to FRET, including insensitivity to spectraloverlap (allowing donor chromophores to excite significantly red-shiftedacceptors), decreased dependence on donor acceptor dipole alignment, andaccelerated energy transfer kinetics (due to the lack of a strictorientational requirement).

Many of the previously described tetrazine-based probes achieve theirfluorogenic turn-on by a unique mechanism, in which the tetrazine (Tz)chromophore is both quencher and bioorthogonal reactant. In publishedstudies, flexibly-linked Tz-fluorophore pairs—chosen for their readysynthetic accessibility—are quenched with moderate efficiency, yieldingturn-on ratios on the order of 10-20 fold after reaction with dienophiletargets. Although intriguing applications have been demonstrated, thelimited turn-on ratios almost always result in native background duringimaging applications. In some cases, a larger turn-on ratio is requiredfor useful imaging. For example, a turn-on ratio of 10² would bepreferable for robust utility in cellular imaging applications, and aratio of 10³ may be necessary for low abundance targets and superresolution imaging. Mechanistic observations have suggested thatquenching in bichromophoric fluorophore-tetrazines occurs via Försterresonance energy transfer (FRET), offering a starting point for effortsto optimize turn-on. Although its relatively weak visible lightabsorbance inherently limits the range of tetrazine as a FRET acceptor,Förster theory dictates that energy transfer efficiency will becrucially dependent upon inter-chromophore distance (varying as r⁶) andupon transition dipole alignment, which are both optimizable parameters.Without being bound by theory, an alternative way of designing moreefficient turn-on probes, involves adapting through-bond energy transfer(TBET) for fluorescence quenching as is illustrated by the compoundsprovided herein.

Accordingly, provided herein are compounds of Formula (I):

F-L-Tz

or a salt thereof,

-   wherein:-   F is a fluorophore;-   L is a conjugated linker; and-   Tz is a substituted or unsubstituted tetrazine;-   wherein the linker bridges the Tz and F moieties in a single    conjugated pi-system.

The single, conjugated pi-system may, in some embodiments, benon-coplanar due to steric factors within the compound that enforce atwist in the interchromophore linkage between the F and L-Tz moieties.Such steric factors can originate from substituents on either the linkeror on the fluorophore. Without being bound by theory, fluorescence canbe increased when the L-Tz moiety is oriented with respect to the Fmoiety such that the tetrazine transition dipole is either collinearwith or parallel to the transition dipole of the fluorophore (see, e.g.,FIG. 8). In some embodiments, the pi-system is topologically conjugation(i.e., a single systems of alternating single and double bonds). Inother embodiments, for example, when the pi-system is coplanar, theconjugation of the pi-system is both topologically and functionally(i.e., wherein the alternating single and double bonds are coplanar)conjugated.

In some embodiments, the compounds provided herein enhance spatialdonor-acceptor proximity, provide predictable donor-acceptor transitiondipole orientation, and/or afford the possibility of accessing alternatemodes of fluorescence quenching.

A “fluorophore”, as described herein, can be any small molecule that canre-emit light upon light excitation (e.g., light in the visiblespectrum). For example, fluorophores can include rhodamines,fluoresceins, boron-dypyrromethanes, coumarins, pyrenes, cyanines,oxazines, acridines, auramine Os, and derivatives thereof. In somecases, derivatives include sulfonated derivatives such as sulfonatedpyrenes, sulfonated coumarins, sulfonated rhodamines, and sulfonatedcyanines (e.g., ALEXAFLUOR® dyes).

In particular, boron-dypyrromethanes fluorophores may includeboron-dipyrromethene (BODIPY®);4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid(BODIPY® FL);6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoicacid, succinimidyl ester (BODIPY® TRM-X);6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid, succinimidyl ester (BODIPY® 650/665-X);5,5-difluoro-1,3,7,9,10-pentamethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide;4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene-8-PropionicAcid;4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-PropionicAcid;4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-PentanoicAcid;6-((4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionyl)amino)hexanoicAcid; 4,4-Difluoro-5-Phenyl-4-Bora-3a,4a-Diaza-s-Indacene-3-PropionicAcid;4,4-Difluoro-5,7-Diphenyl-4-Bora-3a,4a-Diaza-s-Indacene-3-PropionicAcid;6-((4,4-Difluoro-1,3-Dimethyl-5-(4-Methoxyphenyl)-4-Bora-3a,4a-Diaza-s-Indacene-2-Propionyl)amino)hexanoicAcid;4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-PropionicAcid; 4,4-Difluoro-5-Styryl-4-Bora-3a,4a-Diaza-s-Indacene-3-PropionicAcid;4,4-Difluoro-5-(2-Pyrrolyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-PropionicAcid;4,4-Difluoro-5-(4-Phenyl-1,3-Butadienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-PropionicAcid;64(4-(4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-yl)phenoxy)acetyl)amino)hexanoicAcid;6-(((4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-yl)styryloxy)acetyl)aminohexanoicAcid;6-(((4,4-Difluoro-5-(2-Pyrrolyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-yl)Styryloxy)Acetyl)AminohexanoicAcid; 5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoicacid; 5-decyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionicacid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoicacid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoicacid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoicacid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoicacid;4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoicacid; 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoicacid; 4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoicacid;4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoicacid;4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoicacid;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoicacid;2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine;2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine;2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine;2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine;2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine;2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate;N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine;N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine;cholesteryl4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate;cholesteryl4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate;cholesteryl4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate;4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene;4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene;N-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-yl)Methyl)Iodoacetamide;N-(4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene-2-yl)Iodoacetamide;8-Bromomethyl-4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene;or salts and/or ester derivatives thereof. In some embodiments, apharmaceutically acceptable salt includes sodium, diammonium, andtriethylammonium. In some embodiments, a pharmaceutically acceptableester derivative includes succinimidyl ester and sulfosuccinimidyl esterderivatives.

Also contemplated herein are the BODIPY compounds described in Ulrich,G. et al., Angew. Chem. Int. Ed. 2008, 47: 1184-1201; Ziessel, R. etal., New J Chem. 2007, 31: 496-501; and Loudet, A. and Burgess, K.,Chem. Rev. 2007, 107: 4891-4932, Jiang, X-D et al., Org. Letters 201214(1): 248-251; Zhu, S. et al., Org. Letters, 2011 13(3): 438-441; Bura,T. and Ziessel, R., Org. Letters 2011 13(12): 3072-3075; Niu, S. L. etal., Org. Letters 2009 11(10): 2049-2052; Li, L. et al., JOC 2008 73:1963-1970, and Tahtaoui, C. et al., JOC 2007 72: 269-272.

In some cases, BODIPY fluorophores can include a group having thestructure:

or a salt thereof,

-   wherein:-   R¹ and R² are selected from the group consisting of: H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —COR¹⁰, —CO₂R¹⁰, —SOR¹², —SO₂R¹², —NR¹⁰R¹¹, —NO₂,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted, and wherein if both R¹ and R² are present, no more    than one of R¹ and R² is H;-   R³, R⁴, R⁵, R⁶, and R⁷ are independently selected from H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —SO₃H, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10    membered heterocyclyl, 5-10 membered heteroaryl, each of which is    independently substituted or unsubstituted, and a reactive moiety;-   R⁸ and R⁹ are independently selected from halogen (e.g., fluorine),    (C₁-C₆)alkyl, (C₂-C₆)alkynyl, —CO₂R¹⁰, (C₁-C₆)alkoxy, O(4 membered    heterocyclyl), —O—(C₁-C₆)alkyl-O(nPEG), each of which is    independently substituted or unsubstituted;-   each R¹⁰ and R¹¹ are independently selected from H and (C₁-C₆)alkyl;-   each R¹² is independently a (C₆-C₁₀)aryl.

In some embodiments, if one of R¹ or R² is H, the other can be a(C₁-C₆)alkyl, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, or 5-10 memberedheteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl,tert-butyl, or isopropyl. In some embodiments, R¹ and R² areindependently selected from methyl, ethyl, isopropyl, and tert-butyl.

-   In some embodiments, R³, R⁴, R⁵, R⁶, and R⁷ are independently    selected from H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₆-C₁₀)aryl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted. For example, R³, R⁴, R⁵, R⁶, and R⁷ can be    independently selected from H, methyl, ethyl,

R⁸ and R⁹ are independently selected from halogen (e.g., fluorine),(C₂-C₆)alkynyl, —CO₂R¹⁰, (C₁-C₆)alkoxy, O(4 membered heterocyclyl),—O—(C₁-C₆)alkyl-O(nPEG), each of which is independently substituted orunsubstituted. For example, R⁸ and R⁹ can be independently selected fromF, —OCO₂H, —OCH₃,

wherein X is O, N, or S, and nPEG is a polyethylene glycol polymer.

Coumarin fluorophores include, for example, 2H-chromen-2-one,umbelliferone (7-hydroxycoumarin), aesculetin (6,7-dihydroxycoumarin),herniarin (7-methoxycoumarin), psoralen, and imperatorin. In someembodiments, a coumarin fluorophore includes a group selected from:

or a salt thereof,

-   wherein:-   R¹ and R² are selected from the group consisting of: H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —COR¹⁰, —CO₂R¹⁰, —SOR¹², —SO₂R¹², —NR¹⁰R¹¹, —NO₂,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted;-   R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are independently selected from H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —SO₃H, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10    membered heterocyclyl, 5-10 membered heteroaryl, each of which is    independently substituted or unsubstituted, and a reactive moiety;-   each R¹⁰ and R¹¹ are independently selected from H and (C₁-C₆)alkyl;    and-   each R¹² is independently a (C₆-C₁₀)aryl.

In some cases, wherein if both R¹ and R² are present, no more than oneof R¹ and R² is H (i.e., at least one of R¹ and R² is not H). In some ofthese embodiments, if one of R¹ or R² is H, the other can be a(C₁-C₆)alkyl, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, or 5-10 memberedheteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl,tert-butyl, or isopropyl. In some embodiments, R¹ and R² areindependently selected from H, methyl, ethyl, isopropyl, and tert-butyl.In some embodiments, R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹, when present, areH.

In some embodiments, a coumarin fluorophore is selected from the groupconsisting of:

or a salt thereof.For example, a coumarin fluorophore can include:

or a salt thereof.

A fluorophore can be a rhodamine fluorophore. Non-limiting examplesinclude rhodamine, rhodamine B, rhodamine 6G, rhodamine 123,carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and itsisothiocyanate derivative (TRITC), sulforhodamine 101 (and its sulfonylchloride form Texas Red), Rhodamine Red, Alexa 546, Alexa 555, Alexa633, DyLight 550, and DyLight 633.

In some cases, a rhodamine fluorophore can be a group selected from:

or a salt thereof,

-   wherein:-   X is a counter anion;-   R¹ and R² are selected from the group consisting of: H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —COR¹⁴, —CO₂R¹⁴, —SOR¹⁶, —SO₂R¹⁶, —NR¹⁴R¹⁵, —NO₂,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted;-   R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are independently    selected from the group consisting of: H, (C₁-C₆)alkyl,    (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy,    —SO₃, —SO₃H, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered    heterocyclyl, 5-10 membered heteroaryl, each of which is    independently substituted or unsubstituted, and a reactive moiety;    wherein, optionally, one or more of the pairs R³ and R⁷, R⁴ and R⁸,    R⁵ and R⁹, and R⁶ and R¹⁰ come together to form a saturated or    unsaturated ring structure with the carbons or nitrogens to which    they are attached, any of which can be substituted or unsub    stituted;-   each R¹⁴ and R¹⁵ are independently selected from H and (C₁-C₆)alkyl;    and-   each R¹⁶ is independently a (C₆-C₁₀)aryl.

In some cases, wherein if both R¹ and R² are present, no more than oneof R¹ and R² is H (i.e., at least one of R¹ and R² is not H). In some ofthese embodiments, if one of R¹ or R² is H, the other can be a(C₁-C₆)alkyl, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, or 5-10 memberedheteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl,tert-butyl, or isopropyl. In some embodiments, R¹ and R² areindependently selected from H, methyl, ethyl, isopropyl, and tert-butyl.

Fluorescein fluorophores can include fluorescein, fluoresceinisothiocyanate (FITC), 6-FAM phosphoramidite, esters of fluoresceinincluding succinimidyl esters (NHS-fluorescein), pentafluorophenylesters (PFP) and tetrafluorophenyl esters (TFP), eosin,carboxyfluorescein, fluorescein amidite (FAM), merbromin, erythrosine,Rose Bengal, and DyLight Fluor agents. In some embodiments, afluorescein fluorophore is a group selected from:

or a salt thereof,

-   wherein:-   R¹ and R² are selected from the group consisting of: H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —COR¹⁰, —CO₂R¹⁰, —SOR¹², —SO₂R¹², —NR¹⁰R¹¹, —NO₂,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted;-   R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are independently selected from the    group consisting of: H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,    (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₃-C₁₀)carbocyclyl,    (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, 5-10 membered heteroaryl,    each of which is independently substituted or unsubstituted, and a    reactive moiety;-   each R¹⁰ and R¹¹ are independently selected from H and (C₁-C₆)alkyl;    and-   each R¹² is independently a (C₆-C₁₀)aryl.

In some cases, wherein if both R¹ and R² are present, no more than oneof R¹ and R² is H (i.e., at least one of R¹ and R² is not H). In some ofthese embodiments, if one of R¹ or R² is H, the other can be a(C₁-C₆)alkyl, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, or 5-10 memberedheteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl,tert-butyl, or isopropyl. In some embodiments, R¹ and R² areindependently selected from H, methyl, ethyl, isopropyl, and tert-butyl.

A fluorophore, as described herein, can include a combinationfluorophore based on a combination of rhodamine and fluorescein. Forexample, a fluorophore can be selected from:

or a salt thereof,

-   wherein:-   X is a counter anion;-   R¹ and R² are selected from the group consisting of: H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —COR¹², —CO₂R¹², —SOR¹⁴, —SO₂R¹⁴, —NR¹²R¹³, —NO₂,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted;-   R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are independently selected    from the group consisting of: H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,    (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, —SO₃, —SO₃H,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, 5-10    membered heteroaryl, each of which is independently substituted or    unsubstituted, and a reactive moiety; wherein, optionally, one or    more of the pairs R⁵ and R⁶, and R⁷ and R⁸ come together to form a    saturated or unsaturated ring structure with the carbons or    nitrogens to which they are attached, any of which can be    substituted or unsubstituted;-   each R¹² and R¹³ are independently selected from H and (C₁-C₆)alkyl;    and-   each R¹⁴ is independently a (C₆-C₁₀)aryl.

In some cases, wherein if both R¹ and R² are present, no more than oneof R¹ and R² is H (i.e., at least one of R¹ and R² is not H). In some ofthese embodiments, if one of R¹ or R² is H, the other can be a(C₁-C₆)alkyl, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, or 5-10 memberedheteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl,tert-butyl, or isopropyl. In some embodiments, R¹ and R² areindependently selected from H, methyl, ethyl, isopropyl, and tert-butyl.

In some embodiments, a fluorophore can be a group selected from:

or a salt thereof,

-   wherein:-   R¹ and R² are selected from the group consisting of: H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —COR¹², —CO₂R¹², —SOR¹⁴, —SO₂R¹⁴, —NR¹²R¹³, —NO₂,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted;-   R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are independently selected    from the group consisting of: H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,    (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, (C₃-C₁₀)carbocyclyl,    (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, 5-10 membered heteroaryl,    each of which is independently substituted or unsubstituted, and a    reactive moiety; wherein, optionally, one or more of the pairs R⁴    and R⁷, and R⁵ and R⁶ come together to form a saturated or    unsaturated ring structure with the carbons or nitrogens to which    they are attached, any of which can be substituted or unsubstituted;-   each R¹² and R¹³ are independently selected from H and (C₁-C₆)alkyl;    and-   each R¹⁴ is independently a (C₆-C₁₀)aryl.

In some cases, wherein if both R¹ and R² are present, no more than oneof R¹ and R² is H (i.e., at least one of R¹ and R² is not H). In some ofthese embodiments, if one of R¹ or R² is H, the other can be a(C₁-C₆)alkyl, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, or 5-10 memberedheteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl,tert-butyl, or isopropyl. In some embodiments, R¹ and R² areindependently selected from H, methyl, ethyl, isopropyl, and tert-butyl.

A fluorophore can include a pyrene fluorophore. Non-limiting examples ofa pyrene fluorophore include a group selected from:

or a salt thereof,

-   wherein:-   R¹ and R² are selected from the group consisting of: H,    (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,    (C₁-C₆)alkoxy, —COR¹³, —CO₂R¹³, —SOR¹⁵, —SO₂R¹⁵, —NR¹³R¹⁴, —NO₂,    (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and    5-10 membered heteroaryl, each of which is independently substituted    or unsubstituted;-   R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently    selected from the group consisting of: H, (C₁-C₆)alkyl,    (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₆)alkoxy,    —SO₃H, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered    heterocyclyl, 5-10 membered heteroaryl, each of which is    independently substituted or unsubstituted, and a reactive moiety;-   each R¹³ and R¹⁴ are independently selected from H and (C₁-C₆)alkyl;    and-   each R¹⁵ is independently a (C₆-C₁₀)aryl.

In some cases, wherein if both R¹ and R² are present, no more than oneof R¹ and R² is H (i.e., at least one of R¹ and R² is not H). In some ofthese embodiments, if one of R¹ or R² is H, the other can be a(C₁-C₆)alkyl, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, or 5-10 memberedheteroaryl. For example, phenyl, cyclohexyl, pyridyl, cyclopentyl,tert-butyl, or isopropyl. In some embodiments, R¹ and R² areindependently selected from H, methyl, ethyl, isopropyl, and tert-butyl.

Additional fluorophores may also be used in the compounds describedherein, for example, cyanine fluorophores. For example, a fluorophorehaving the structure:

or a salt thereof. In some embodiments, the fluorophore can be anoxazine fluorophore. For example, the fluorophore can be selected from:

or a salt thereof, wherein X is a counter anion as described above. Afluorophore can also be an acridine fluorophore. For example, thefluorophore can be a compound having the strucuture:

or a salt thereof. In some embodiments, a fluorophore can be an auramineO fluorophore. For example, a fluorophore can be a compound:

or a salt thereof. As for the fluorophores described previously, thelocation of attachment of the -L-Tz moiety may vary and the fluorophoresmay be further substituted to modify the steric and/or fluorescentcharacteristics of the compound.

A linker moiety can be a conjugated linker. The linker containsalternating single and multiple bonds and can be cyclic, acyclic,linear, or any combination thereof. For example, the linker can includean alkenyl, alkynyl, aryl, and/or heteroaryl moiety. In someembodiments, the linker can include an alkenyl and an aryl, an alkynyland an aryl, an alkenyl and a heteroaryl, or an alkynyl and aheteroaryl. The structure of the linker must be such that the linkerbridges and continues the conjugation of the fluorophore with that ofthe tetrazine so that a conjugated pi-system extends between these threemoieties.

In some embodiments, a linker moiety can be an aromatic linker. Forexample, the aromatic linker can be a (C₆-C₁₀)aryl or 5-10 memberedheteroaryl. In some embodiments, the aromatic linker is selected fromthe group consisting of:

wherein:

-   R^(1a), R^(2a), R^(3a), and R^(4a) are selected from the group    consisting of: H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl,    (C₂-C₆)alkynyl, (C₁-C₆)alkoxy, —COR¹³, —CO₂R¹³, —SOR¹⁵, —SO₂R¹⁵,    —NR¹³R¹⁴, —NO₂, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered    heterocyclyl, and 5-10 membered heteroaryl, each of which is    independently substituted or unsubstituted.    Additional examples of aromatic linkers include:

In some embodiments, a linker is

wherein the linker is substituted by F and Tz as exemplified above. Asabove, the linkers described herein can be further substituted to modifythe sterics of the linker and the orientation of the L-Tz moiety withrespect to the F moiety.

Linkers, as provided herein, can also be selected from a substituted orunsubstituted heteroaryl or heterocyclyls. Non-limiting examplesinclude:

each of the above compounds is independently substituted by F and Tz(e.g., through a carbon-carbon bond). In addition, each of the compoundsdescribed above may be unsubstituted or substituted (e.g., by one ormore R_(x) as described below). In some embodiments, the heteroaryl orheterocyclic linkers provided herein can include an alkenyl or alkynyllinkage between the fluorophore and/or the tetrazine moiety.

In some embodiments, a linker can be selected from the group consistingof:

wherein n is an integer from 1 to 10 (e.g., 1 to 4); and each Rx isindependently selected from the group consisting of H, (C₁-C₆)alkyl,(C₁-C₆)alkoxyl, and a tetrazine moiety as provided herein. For example,each R^(x) can be independently selected from H, methyl, ethyl, propyl,isopropyl, methoxy, and a tetrazine moiety as provided herein.

The tetrazine moiety in the compound of Formula (I) can be a substitutedor unsubstituted tetrazine. In some embodiments, the tetrazine moiety istetrazine. In other embodiments, the tetrazine is substituted with asubstituent selected from Cy¹, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,halo, C₁₋₆ haloalkyl, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),C(═NR^(e1))NR^(c1)R^(d1), NR^(c1)C(═NR^(e1))NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1),NR^(c1)C(O)NR^(c1)R^(d1), NR^(c1)S(O)R^(b1), NR^(c1)S(O)₂R^(b1),NR^(c1)S(O)₂NR^(c1)R^(d1), S(O)R^(b1), S(O)NR^(c1)R^(d1), S(O)₂R^(b1)and S(O)₂NR^(c1)R^(d1), wherein

each Cy¹ is independently C₆₋₁₀ aryl, C₃₋₁₀ carbocyclyl, 5-10 memberedheteroaryl or 4-10 membered heterocyclyl, each of which is unsubstitutedor substituted by 1, 2, 3, 4 or 5 substituents independently selectedfrom C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), C(═NR^(e1))NR^(c1)R^(d1),NR^(c1)C(═NR^(e1))NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), NR^(c1)C(O)NR^(c1)R^(d1), NR^(c1)S(O)R^(b1),NR^(c1)S(O)₂R^(b1), NR^(c1)S(O)₂NR^(c1)R^(d1), S(O)R^(b1),S(O)NR^(c1)R^(d1), S(O)₂R^(b1), S(O)₂NR^(c1)R^(d1) and oxo;

each R^(a1), R^(b1), R^(c1), R^(d1) is independently selected from H,C₁₋₆ alkyl, C₁₋₄ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₆₋₁₀ aryl,C₃₋₁₀ carbocyclyl, 5-10 membered heteroaryl, 4-10 membered heterocyclyl,C₆₋₁₀ aryl-C₁₋₄ alkyl, C₃₋₁₀ carbocyclyl-C₁₋₄ alkyl, (5-10 memberedheteroaryl)-C₁₋₄ alkyl or (4-10 membered heterocyclyl)-C₁₋₄ alkyl.

For example, a tetrazine can be a moiety having the structure:

wherein:

-   R^(1b) is selected from the group consisting of: H, (C₁-C₆)alkyl,    (C₁-C₆)haloalkyl, (C₆-C₁₀)aryl, and 5-10 membered heteroaryl, each    of which is independently substituted or unsubstituted.    For example, the tetrazine can be selected from:

Non-limiting examples of a compound of Formula (I) include:

or a salt thereof, wherein each R is independently selected from H,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₆-C₁₀)aryl, and 5-10 memberedheteroaryl. In some embodiments, R is H or (C₁-C₆)alkyl, such as CH₃.

For example, a compound of Formula (I) can include:

or a salt thereof.

Also provided herein is a compound of Formula (II):

F-Tz

or a salt thereof, wherein F is a fluorophore and Tz is a substituted orunsubstituted tetrazine as described previously, and wherein the Tz andF moieties form a single conjugated pi-system. In some embodiments, theTz and F moieties form a non-coplanar pi-system. For example, the Tzmoiety can be oriented with respect to the F moiety such that thetetrazine transition dipole is either collinear with or parallel to thetransition dipole of the fluorophore. The moieties F and Tz can be asdefined for any of the embodiments described above.

Without being bound by any theory, the most TBET quenched probe may beone where the tetrazine is directly attached to the fluorophore. Innon-planar compounds, the disruption in conjugation between the twosystems can be provided by the fluorophore (e.g., by the steric effectsof substituents on the fluorophore). In effect, the TBET connectionbetween the two systems is the shortest possible (i.e., the single bondbetween the two moieties).

Non-limiting examples of a compound of Formula (II) include:

or a salt thereof.

This disclosure further provides a compound of Formula (III):

F-L-Z

or a salt thereof,

-   wherein:-   F is a fluorophore;-   L is a conjugated linker; and-   Z is a moiety comprising the reaction product of a diene and a    dienophile, wherein the diene is a tetrazine or a derivative    thereof;-   wherein the linker bridges the Z and F moieties in a single    conjugated pi-system.

The single, conjugated pi-system may, in some embodiments, benon-coplanar due to steric factors within the compound that enforce atwist in the interchromophore linkage between the F and L-Z moieties.Such steric factors can originate from substituents on either the linkeror on the fluorophore. Without being bound by theory, fluorescence canbe increased when the L-Z moiety is oriented with respect to the Fmoiety such that the transition dipole of Z is either collinear with orparallel to the transition dipole of the fluorophore.

The fluorophore, linker, and tetrazine are as described above.

Z is a moiety formed through the reaction of a diene (e.g., tetrazine ora derivative thereof) and a dienophile using bioorthogonal chemistry.Bioconjugation methods using inverse electron demand Diels-Aldercycloadditions between tetrazines and highly strained dienophiles suchas norbornene, strained cyclic alkenes, and trans-cyclooctene are knownin the literature, however the tetrazine used has limited stability toaqueous media. (Blackman et al., 2008, J Am Chem Soc, 130, 13518-9;Devaraj et al., 2009, Angew Chem Int Ed Engl, 48, 7013-6; Devaraj etal., 2008, Bioconjug Chem, 19, 2297-9; Pipkorn et al., 2009, J Pept Sci,15, 235-41).

Dienophiles useful in the compounds described herein include but are notlimited to carbon containing dienophiles such as alkenes or alkynes, orcompounds containing nitroso, carbonyl or imine groups. In someembodiments, the dienophile is a strained dienophile. As used herein, a“strained” dienophile has a dihedral angle that deviates from theidealized 180 degree dihedral angle. Alternatively, non-straineddienophiles (e.g., styrenes) and/or electron rich electrophiles (e.g.,eneamines or vinyl ethers), can also be used with nitroso compounds.Alkenes as used herein refers to an alkyl group having one or moredouble carbon-carbon bonds such as an ethylene, propylene, and the like.Alkenes can also include cyclic, ring-strained alkenes such astrans-cyclooctene or norbornene carrying a double bond which inducessignificant ring strain and is thus highly reactive. Alkenes can alsoinclude more complex structures such as indoles and azaindoles, electronrich enamines. Heterodienophiles containing carbonyl, nitroso or iminegroups can also be used. In some embodiments, the dienophile is atrans-cyclooctenol, e.g., (E)-cyclooct-4-enol. Additional examples ofdienophiles include those described in Thalhammer, F. et al. TetrahedronLetters 1990 31(47): 6851-6854. In some embodiments, the dienophile is asubstituted or derivatized trans-cyclooctenol. For example, the hydroxylmoiety on the trans-cyclooctenol can be derivatized with a linker (e.g.,a PEG linker) and/or a reactive moiety. In some embodiments, thetrans-cyclooctenol is modified with a reactive moiety such as:

wherein n is an integer from 1 to 10; or a carbamate-linked PEG (e.g., acarbamate-linked PEGm, wherein m is an integer from 1 to 10) such as acarbamate-linked PEG₂ (TCOc):

Non-limiting examples of a compound of Formula (III) include:

or a salt thereof.

Also provided herein is a compound of Formula (IV):

F-Z

or a salt thereof, wherein F is a fluorophore and Z is a moietycomprising the reaction product of a diene and a dienophile, wherein thediene is a tetrazine or a derivative thereof as described previously,and wherein the Z and F moieties form a single conjugated pi-system.

The compounds provided herein can be referred to as Hyper EmissiveLigation-Initiated Orthogonal Sensing (HELIOS) probes. Many of thecompounds provided herein are designated HELIOS XXX, where XXX refers tothe absorption maximum of the “turned on” probe. This designation isused to refer to certain compounds within the present disclosure.

Synthesis

The compounds provided herein, including salts thereof, can be preparedusing known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes.

The reactions for preparing the compounds provided herein can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of the compounds provided herein can involve the protectionand deprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in Protecting GroupChemistry, 1^(st) Ed., Oxford University Press, 2000; March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, 5^(th) Ed.,Wiley-Interscience Publication, 2001; and Peturssion, S. et al.,“Protecting Groups in Carbohydrate Chemistry,” J. Chem. Educ., 74(11),1297 (1997).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC), liquid chromatography-mass spectroscopy(LCMS), or thin layer chromatography (TLC). Compounds can be purified bythose skilled in the art by a variety of methods, including highperformance liquid chromatography (HPLC) (“Preparative LC-MSPurification: Improved Compound Specific Method Optimization” K. F.Blom, et al., J. Combi. Chem. 6(6), 874 (2004) and normal phase silicachromatography.

In some embodiments, the compounds provided herein can be prepared asdescribed in the Examples provided herein and as illustrated in Scheme1.

As another example, a compound as provided herein can also be preparedas illustrated in Scheme II.

As another example, a compound as provided herein can also be preparedas illustrated in Scheme III.

Pharmaceutical Compositions

Pharmaceutical compositions can include any of the compounds describedherein, and can be formulated as a pharmaceutical composition inaccordance with routine procedures. As used herein, pharmaceuticalcompositions can include pharmaceutically acceptable salts orderivatives thereof. “Pharmaceutically acceptable” means that the agentcan be administered to an animal without unacceptable adverse effects. A“pharmaceutically acceptable salt or derivative” means anypharmaceutically acceptable salt, ester, salt of an ester, or otherderivative of composition that, upon administration to a recipient, iscapable of providing (directly or indirectly) a composition of thepresent disclosure. Other derivatives are those that increase thebioavailability when administered to a mammal (e.g., by allowing anorally administered compound to be more readily absorbed into the blood)or which enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) thereby increasing theexposure relative to the parent species. Pharmaceutically acceptablesalts of the therapeutic or diagnostic compositions or compositions ofthis disclosure include counter ions derived from pharmaceuticallyacceptable inorganic and organic acids and bases known in the art, e.g.,sodium, calcium, N-methylglutamine, lithium, magnesium, potassium, etc.

Pharmaceutical compositions can be administered by any route, includingoral, intranasal, inhalation, or parenteral administration. Parenteraladministration includes, but is not limited to, subcutaneous,intravenous, intraarterial, interstitial, intrathecal, and intracavityadministration. When administration is intravenous, pharmaceuticalcompositions may be given as a bolus, as two or more doses separated intime, or as a constant or non-linear flow infusion. Thus, compositionscan be formulated for any route of administration.

Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent, a stabilizing agent, and a localanesthetic such as lidocaine to ease pain at the site of the injection.Generally, the ingredients will be supplied either separately, e.g. in akit, or mixed together in a unit dosage form, for example, as a drylyophilized powder or water free concentrate. The composition may bestored in a hermetically sealed container such as an ampule or sachetteindicating the quantity of active agent in activity units. Where thecomposition is administered by infusion, it can be dispensed with aninfusion bottle containing sterile pharmaceutical grade “water forinjection,” saline, or other suitable intravenous fluids. Where thecomposition is to be administered by injection, an ampule of sterilewater for injection or saline may be provided so that the ingredientsmay be mixed prior to administration. Pharmaceutical compositionscomprise the therapeutic or diagnostic compositions of the presentdisclosure and pharmaceutically acceptable salts thereof, with anypharmaceutically acceptable ingredient, excipient, carrier, adjuvant orvehicle.

A pharmaceutical composition is preferably administered to the subjectin the form of an injectable composition. The method of administering atherapeutic or diagnostic composition is preferably parenterally,meaning intravenously, intra-arterially, intrathecally, interstitiallyor intracavitarilly. Pharmaceutical compositions can be administered tomammals including humans in a manner similar to other diagnostic ortherapeutic agents. The dosage to be administered, and the mode ofadministration will depend on a variety of factors including age,weight, sex, condition of the subject and genetic factors, and willultimately be decided by medical personnel subsequent to experimentaldeterminations of varying dosage followed by imaging as describedherein. In general, dosage required for diagnostic sensitivity ortherapeutic efficacy will range from about 0.001 to 50,000 μg/kg,preferably between 0.01 to 25.0 μg/kg of host body mass. The optimaldose will be determined empirically following the disclosure herein.

Methods of Use

The compounds described herein can be imaged using methods known in theart. For example, a compound provided herein can be detected bytraditional fluorescence imaging techniques allowing for the faciletracking of the compounds by fluorescence microscopy or flow cytometryusing methods known in the art, e.g., as described in US 2005/0249668.

The compositions and methods described herein can be imaged using avariety of modalities that are known to one of skill in the art.Detection methods can include both imaging ex vivo and in vivo imagingmethods, e.g., fluorescence reflectance imaging, fluorescencemicroscopy, super-resolution microscopy (see, e.g., M. Bates et al.,Science 2007, 317: 1749; and G. Patterson et al., Annu. Rev. Phys. Chem.2010, 61: 345), and fluorescence molecular tomographic imaging. In someembodiments, one or more imaging techniques can be used in the methodsprovided herein.

After a compound has been designed, synthesized, and optionallyformulated, it can be tested in vitro by one skilled in the art toassess its biological and performance characteristics. For instance,different types of cells grown in culture can be used to assess thebiological and performance characteristics of the compound. Cellularuptake, binding or cellular localization of the agent can be assessedusing techniques known in the art, including, for example, fluorescentmicroscopy and fluorescence-activated cell sorting (FACS) analysis.

By way of example, the compound can be contacted with a sample for aperiod of time. The sample can then be viewed using an appropriatedetection device without the need for washing or clearance steps. Adetection device can include a fluorescent microscope equipped withappropriate filters matched to the optical properties of a fluorescentagent. Fluorescence microscopy of cells in culture is also a convenientmeans for determining whether uptake and binding has occurred. Tissues,tissue sections and other types of samples can also be used in a similarmanner to assess the biological and performance characteristics of thecompounds.

Formation of a compound of Formula (III) can occur through the reactionof a compound of Formula (I) with the corresponding dienophile.Similarly, the formation of a compound of Formula (IV) can occur throughthe reaction of a compound of Formula (II) with the correspondingdienophile.

In some embodiments, a compound of Formula (I) or (II) exhibits low orno fluorescence prior to activation upon reaction with a dienophile. Forexample, the turn-on ratio of a compound of Formula (III) or (IV) (i.e.,the reaction product of the corresponding compound of Formula (I) or(II) with the corresponding dienophile) can be greater than 50 (e.g.,greater than 60, greater than 75, greater than 100, greater than 125,greater than 150, greater than 200, greater than 300, greater than 400,greater than 500, greater than 600, greater than 700, greater than 800,greater than 900, greater than 1000, greater than 1500, greater than2000, greater than 2500, greater than 3000, greater than 3500, greaterthan 4000, greater than 4500, greater than 5000, greater than 5500,greater than 6000, greater than 6500, greater than 7000, greater than7500, greater than 8000, greater than 8500, greater than 9000, greaterthan 9500, greater than 10,000, greater than 11,000, greater than12,000, greater than 14,000, greater than 15,000, greater than 16,000,greater than 18,000, greater than 20,000, greater than 22,000, greaterthan 24,000, greater than 26,000, greater than 28,000, greater than30,000). In some embodiments, the turn-on ratio of a compound of Formula(III) or (IV) can range from about 100 to about 2000 (e.g., from about100 to about 1800; from about 100 to about 1600; from about 100 to about1250; from about 100 to about 1000; from about 100 to about 800; fromabout 100 to about 750; from about 100 to about 500; from about 100 toabout 400; from about 100 to about 300; from about 200 to about 2000;from about 400 to about 2000; from about 600 to about 2000; from about800 to about 2000; from about 1000 to about 2000; from about 1200 toabout 2000; from about 1500 to about 2000; from about 200 to about 1800;from about 400 to about 1600; from about 500 to about 1500; and fromabout 750 to about 1250). In some embodiments, the turn-on ratio of acompound of Formula (III) or (IV) can range from about 1000 to about30,000 (e.g., from about from about 1000 to about 25,000; from about1000, to about 22,000; from about 1000 to about 18,000; from about 1000to about 16,000; from about 1000 to about 12,500; from about 1000 toabout 10,000; from about 1000 to about 8,000; from about 1000 to about7,500; from about 1000 to about 5000; from about 1000 to about 4000;from about 1000 to about 3000; from about 2000 to about 30,000; fromabout 4000 to about 30,000; from about 6000 to about 30,000; from about8000 to about 30,000; from about 10,000 to about 30,000; from about12,000 to about 30,000; from about 15,000 to about 30,000; from about20,000 to about 30,000; from about 2000 to about 12,000; from about4,000 to about 16,000; from about 2500 to about 17,500; from about fromabout 10,000 to about 20,000; from about 5000 to about 15,000; and fromabout 7,500 to about 12,500). Such low or no fluorescence prior toactivation can lead to a background signal which is zero or very nearthe detection limit of the fluorescence instrument.

The bioorthogonal inverse electron demand Diels-Alder reaction can betailored to provide a straightforward method for the rapid, specificcovalent labeling and imaging with ligands such as small molecules andother biomolecules inside living cells. Despite numerous developments inthe application of various selective chemistries to extracellular livecell labeling, to date, no method has been universally adapted tointracellular labeling. For example, described herein are a series of“turn-on” tetrazine-linked fluorescent probes that react rapidly via aninverse electron demand Diels-Alder reaction to strained dienophilessuch as trans-cyclooctene. Upon cycloaddition, the fluorescenceintensity increases dramatically, in some cases by ˜20 fold. Thisfluorescence “turn-on” significantly lowers background signal. Thesenovel probes for live cell imaging of a ligand such as an antibody,small molecule, or other biomolecule modified with a strained alkene canprovide a general method for labeling and imaging a ligand bound to aspecific target. For example, this bioorthogonal inverse electron demandDiels-Alder reaction can be applied to an asymmetric tetrazine and astrained alkene, which is physically coupled to a small molecule, i.e. atrans-cyclooctene modified taxol analog and can be used to label andimage this small molecule bound to intracellular tubules. The rapidreaction rate coupled with fluorescence “turn-on” makes this a nearlyideal method for revealing small molecules inside living cells.

In some embodiments, the ligand, e.g., an antibody, small moleculetherapeutic agent (e.g., a drug), nanoparticles, polymers, or otherbiomolecule, is physically attached (conjugated) to the dienophile. Insome embodiments, the ligand carries a functional group such as anamine, alcohol, carboxylic acid or ester, or other group of atoms on theligand that can undergo a chemical reaction allowing attachment to thedienophile. Alternatively or in addition, the dienophile possesses areactive functional group for attachment to the ligand. Thus, thereactive functional group on the ligand and/or dienophile undergoes achemical reaction to form a link between the two. In some embodiments,e.g., where the ligand is a biopolymer such as a nucleic acid, peptide,or polypeptide, the functional group on the ligand can be a non-naturalnucleoside or amino acid, e.g., as described in Xie and Schultz, Nat.Rev. Mol. Cell Biol. 7:775-782 (2006); for example, the diene ordienophile can be incorporated into a non-natural amino acid as the sidechain. One of skill in the art could readily synthesize such compounds.For example, a compound as provided herein can be modified to include areactive moiety (A):

Non-limiting examples of the reactive moiety (A) include a reactiveester (e.g., NHS ester), a primary amine, carboxylic acid, azide,alkyne, a second tetrazine, maleimide, and thiol. The reactive moietycan be used to couple the compound to additional groups, including smallmolecules (e.g., drugs and therapeutic agents) and nanoparticles. In thecase of a small molecule drug, the fluorescent compounds provided hereincould be used to monitor delivery of the drug following administration.

In some embodiments, a compound as provided herein is imaged in vivousing fluorescent imaging. For example, the use of such methods permitsthe facile, real-time imaging and localization of cells or tissueslabeled with a compound provided herein. In some embodiments, a compoundprovided herein is imaged in vivo using laparoscopy and/orendomiscroscopy. For example, the use of laparoscopy permits the facile,real-time imaging and localization of cells or tissues labeled with acompound provided herein. In some embodiments, a compound can be imagedusing fiber optic endomicroscopy.

A number of preclinical and clinical applications for a compoundprovided herein can be envisioned. For example, a compound describedhere can be used: 1) for the early detection cancers; 2) as an aid tosurgeons during surgery (e.g., by allowing for real-time detection ofcancer cells); and 3) as a method for monitoring the progress of acancer treatment (e.g., by quantifying the cancer cells present before,during, and after treatment).

For example, the compounds provided herein can be administered to asubject in combination with surgical methods, for example, resection oftumors. The compounds can be administered to the individual prior to,during, or after surgery. The compounds can be administeredparenterally, intravenous or injected into the tumor or surrounding areaafter tumor removal, e.g., to image or detect residual cancer cells. Forexample, the compound may be used to detect the presence of a tumor andto guide surgical resection. In some embodiments, the compound can beused to detect the presence of residual cancer cells and to guidecontinued surgical treatment until at least a portion (e.g., all) suchcells are removed from the subject. Accordingly, there is provided amethod of guided surgery to remove at least a portion of a tumor from asubject comprising providing a compound provided herein; causing thecompound to be present in at least some cancer cells; observing theimage following activation of the compound (e.g., fluorescence); andperforming surgery on the subject to remove at least a portion of thetumor that comprises detected cancer cells.

With respect to in vitro imaging methods, the compounds and compositionsdescribed herein can be used in a variety of in vitro assays. Anexemplary in vitro imaging method comprises: contacting a sample, forexample, a biological sample (e.g., a cell), with one or more compoundsprovided herein; allowing the compound to interact with a biologicaltarget in the sample; illuminating the sample with light of a wavelengthabsorbable by a fluorophore of the agents; and detecting a signalemitted from fluorophore thereby to determine whether the agent has beenactivated by or bound to the biological target. In some embodiments, thecompounds provided herein may be used without requiring clearance ofunbound fluorophore prior to imaging.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

General Methods Materials and Methods

All reagents were purchased from Sigma-Aldrich and used without furtherpurification unless otherwise noted. Proton and carbon nuclear magneticresonance (¹H & ¹³C NMR) spectra were recorded on either a Varian AS-400(400 MHz) spectrometer or a Varian 500 MHz spectrometer. Silica Gel 60(40-63 μm) was used for purification. High performance liquidchromatography-mass spectrometry analysis (HPLC-MS) was performed withon a Waters instrument equipped with a Waters 2424 ELS Detector, Waters2998 UV-Vis Diode array Detector, Waters 2475 Multi-wavelengthFluorescence Detector, and a Waters 3100 Mass Detector. Separationsemployed Waters XTerra RP C₁₈ 5 μm or Waters Atlantis RP 5 μm columns,with a water:acetonitrile solvent gradient (0.1% formic acid added).Fluorescence measurements were conducted with a Perkin Elmer LS50BLuminescence Spectrometer, and UV-VIS absorption spectra on an AgilentTechnologies Cary 100 UV-Vis Spectrophotometer.

Fluorescence Assays

The purity of all compounds was verified by LCMS prior to quantitativeactivation experiments and a fresh aliquot of the fluorophore collectedfrom the analytical HPLC elution. Stock solutions in acetonitrile werediluted into 2 mL or 3 mL of the appropriate solvent in a 1 cm×1 cmquartz cuvette. Measurements of solvent and pre-activation emissionspectra for baseline values were made in at least triplicate, prior toaddition of transcyclooctenol (TCO) to initiate the fluorogenicreaction. Activation ratios were calculated from the peak emissionintensity of the reacted dihydropyridazine product and the correspondingbaseline intensity. Integration of the area under the emission intensitycurves was used to validate the activation ratios. For the time-courseexperiments (FIG. 2), the normalized baseline was calculated from themean emission intensity during 30-60 seconds of observation of thesolvent blank followed by the pre-TCO fluorophore solution. Afteraddition of TCO, the fluorescence emission intensity was monitored untila plateau was reached. The rate of activation of 5b was attenuatedrelative to the other methyltetrazines (2b,4b); a 2.5-fold additionalexcess of TCO was added for facile comparison of the magnitude offluorescence turnon.

For quantum yield determinations, fluorescein in 0.1M NaOH was used as areference, with an excitation wavelength of 470 or 480 nm (ex slit 2.5nm); a value of 0.925 was assigned to the quantum yield of fluorescein(Magde, D., Wong, R., & Seybold, P. G. Photochemistry and Photobiology,2002, 75(4), 327-334), and calculations made according to the methodsdescribed by Crosby and Demas (Chemical Reviews, 1971, 75(8), 991-1024).

Example 1 Preparation of Compounds 2a and 2b

To p-cyanophenyl-BODIPY 1 (100 mg, 0.28 mmol) in a microwave reactiontube under a stream of argon was added Zn(OTf)₂ (52.3 mg, 0.14 mmol),MeCN (0.16 mL, 3.0 mmol), DMF (0.34 mL) and NH₂NH₂ (0.55 mL, 16.0 mmol).The vessel was sealed and allowed to stir at 60° C. for 24 hours afterwhich it was allowed to cool and the septum removed. To the reactionmixture was added NaNO₂ (300 mg, 4.34 mmol) in 10 mL of water followedby 1 M HCl until the pH=3. The aqueous phase was extracted three timeswith CH₂Cl₂ (100 mL). The combined organic extracts were dried withMgSO₄ and concentrated using a rotary evaporator. The crude mixture waspurified using flash column chromatography (toluene:hexane gradient 3:1,4:1, 100% toluene) to give 2a and 2b. Compounds 2a and 2b were furtherpurified using flash column chromatography (hexanes:ethyl acetategradient 6:1, 4:1) to give H-tetrazine 2a (6.2 mg, 0.015 mmol, 5.5%) andMe-tetrazine 2b (9.5 mg, 0.022 mmol, 8.1%) as dark red solids. In sharpcontrast to the parent compound 1, both 2a and 2b are modestly solublein aqueous solution and almost completely non-fluorescent.

Compound 2a: ¹H NMR (400 MHz, CD2Cl2) δ 10.29 (s, 1H), 8.80 (d, J=8.4Hz, 2H), 7.64 (d, J=8.0 Hz, 2H), 6.07 (s, 2H), 2.55 (s, 6H), 1.50 (s,6H); ¹³C NMR (100 MHz, CD₂Cl₂) δ 166.1, 158.1, 155.9, 143.1, 140.3,139.8, 132.6, 130.8, 129.4, 128.9, 121.4, 14.4 (4C). ESIMS [M+H]+ calcdfor C₂₁H₂₀BF₂N₆ 405.18, found 405.23.

Compound 2b: ¹H NMR (400 MHz, CD2Cl2) δ 8.76 (d, J=8.8 Hz, 2H), 7.61 (d,J=8.8 Hz, 2H), 6.10 (s, 2H), 3.11 (s, 3H), 2.54 (s, 6H), 1.50 (s, 6H);¹³C NMR (100 MHz, CD2Cl2) δ 167.7, 163.7, 155.9, 143.2, 140.5, 139.1,132.8, 130.9, 129.2, 128.5, 121.4, 21.0, 14.3 (4C). ESIMS [M+H]+ calcdfor C₂₂H₂₂BF₂N₆ 419.20, found 419.29.

Example 2 Preparation of Compound 3

To 3-formylbenzonitrile (1.0 g, 7.6 mmol) and 2,4-dimethylpyrrole (1.7mL, 16.5 mmol) in 200 mL of CH₂Cl₂ under argon atmosphere was added fourdrops of TFA and allowed to stir at room temperature. After 30 minutesthin layer chromatography showed the disappearance of3-formylbenzonitrile after which2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (1.73 g, 7.6 mmol) inCH₂Cl₂ (200 mL) was added followed by N,N-diisopropylethylamine (15.5mL, 88.9 mmol) and BF₃.OEt₂ (15.5 mL, ˜45% BF₃ content). The reactionwas allowed to stir overnight after which water (100 mL) was added andthe aqueous phase was extracted three times with CH₂Cl₂ (300 mL). Thecombined organic extracts were dried with MgSO₄ and concentrated using arotary evaporator. The crude mixture was purified using flash columnchromatography (toluene:hexane gradient 3:1, 100% toluene) to give 3(822.7 mg, 2.3 mmol, 30.2%) as a red solid. ¹H NMR (500 MHz, (CD₃)₂SO) δ8.04 (m, 1H), 7.98 (m, 1H), 7.75 (m, 2H), 6.19 (s, 2H), 2.45 (s, 6H),1.32 (s, 6H)); ¹³C NMR (125 MHz, (CD3)2SO) δ 155.9, 144.5, 143.0, 139.5,135.6, 133.7, 133.5, 132.2, 131.0, 122.2, 121.1, 112.9, 14.7 (4C). ESIMS[M+H]+ calcd for C₂₀H₁₉BF₂N₃ 350.16, found 350.22.

Example 3 Preparation of Compounds 4a and 4b

To 3 (100 mg, 0.28 mmol) in a microwave reaction tube under a stream ofargon was added Zn(OTf)₂ (52.3 mg, 0.14 mmol), MeCN (0.16 mL, 3.0 mmol),DMF (0.34 mL) and NH₂NH₂ (0.55 mL, 16.0 mmol). The vessel was sealed andallowed to stir at 60° C. for 24 hours after which it was allowed tocool and the septum removed. To the reaction mixture was added NaNO₂(300 mg, 4.34 mmol) in 10 mL of water followed by 1 M HCl until thepH=3. The aqueous phase was extracted three times with CH₂Cl₂ (100 mL).The combined organic extracts were dried with MgSO₄ and concentratedusing a rotary evaporator. The crude mixture was purified using flashcolumn chromatography (toluene:acetonitrile gradient 100% toluene,100:0.5) to give 4a and 4b as dark orange solids. Compounds 4a and 4bwere further purified using flash column chromatography (hexanes:ethylacetate 4:1) to give H-tetrazine 4a (9.6 mg, .023 mmol, 8.4%) andMetetrazine 4b (10.1 mg, 0.024 mmol, 8.6%) as dark red solids.

Compound 4a: ¹H NMR (500 MHz, (CD₃)₂SO) δ 10.63 (s, 1H), 8.67 (d, J=8.0Hz, 1H), 8.40 (s, 1H), 7.89 (t, J=8.0 Hz, 1H), 7.78 (d, J=8.0 Hz, 1H),6.21 (s, 2H), 2.47 (s, 6H), 1.41 (s, 6H); ¹³C NMR (125 MHz, (CD₃)₂SO) δ165.6, 158.7, 155.8, 143.1, 140.9, 135.5, 133.6, 132.7, 131.1, 131.0,129.0, 127.6, 122.1, 14.8 (4C). ESIMS [M+H]+ calcd for C₂₁H₂₀BF₂N₆405.18, found 405.27.

Compound 4b: ¹H NMR (500 MHz, (CD₃)₂SO) δ 8.64 (dt, J=8.0, 1.5 Hz, 1H),8.37 (t, J=2.0 Hz, 1H), 7.87, (J=7.5 Hz, 1H), 7.75 (dt, J=7.5, 1.5 Hz,1H), 6.21 (s, 2H), 3.00 (s, 3H), 2.47 (s, 6H), 1.40 (s, 6H); ¹³C NMR(125 MHz, (CD₃)₂SO) δ 167.8, 163.4, 155.8, 143.0, 140.9, 135.5, 133.6,132.3, 132.2, 132.0, 129.1, 127.2, 122.1, 21.3, 14.8 (4C). ESIMS [M+H]+calcd for C₂₂H₂₂BF₂N₆ 419.20, found 419.27.

Example 4 Preparation of Compound 6

To 2-iodo-pentamethyl BODIPY (800 mg, 2.06 mmol) dissolved in 60 mL oftoluene:methanol (5:1) under argon atmosphere was added Na₂CO₃ (874 mg,824 mmol), Pd(OAc)₂ (46.2 mg, 0.20 mmol),2-methyl-4-cyanophenylboronicacid (994.8 mg, 6.2 mmol) and2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl (121.6 mg, 0.31mmol). The mixture was heated to 100° C. and stirred. After 2.5 hours,thin layer chromatography showed the disappearance of 2-iodo-pentamethylBODIPY and the reaction was diluted with 100 mL of water. The aqueousphase was extracted three times with toluene (300 mL). The combinedorganic extracts were dried with MgSO₄ and concentrated using a rotaryevaporator. The crude mixture was purified using flash columnchromatography (toluene) to give 6 (606.3 mg, 1.6 mmol, 77.7%) as a redsolid.

¹H NMR (400 MHz, CD₂Cl₂) δ 7.34 (s, 1H), 7.57 (d, J=7.6 Hz, 1H), 7.25(d, J=7.6 Hz, 1H), 6.17 (s, 1H), 2.67 (s, 3H), 2.52 (s, 3H), 2.48 (s,3H), 2.28 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H); ¹³C NMR (100 MHz, CD₂Cl₂)δ 154.9, 150.2, 142.5, 142.4, 139.6, 138.9, 137.1, 133.5, 132.6, 131.9,131.6, 130.8, 129.4, 121.8, 118.9, 111.5, 19.4, 17.2, 16.7, 15.0, 14.2,12.7. ESIMS [M+H]+ calcd for C₂₂H₂₃BF₂N₃ 378.20, found 378.27.

Example 5 Preparation of Compounds 5a and 5b

To 6 (100 mg, 0.26 mmol) in a microwave reaction tube under a stream ofargon was added Zn(OTf)₂ (49.5 mg, 0.13 mmol), MeCN (0.15 mL, 2.8 mmol),DMF (0.32 mL) and NH₂NH₂ (0.52 mL, 15.1 mmol). The vessel was sealed andallowed to stir at 60° C. for 24 hours after which it was allowed tocool and the septum removed. To the reaction mixture was added NaNO₂(283.8 mg, 4.1 mmol) in 10 mL of water followed by 1 M HCl until thepH=3. The aqueous phase was extracted three times with CH₂Cl₂ (100 mL).The combined organic extracts were dried with MgSO₄ and concentratedusing a rotary evaporator. The crude mixture was purified using flashcolumn chromatography (toluene:acetonitrile gradient 100% toluene,100:0.2) to give 5a and 5b. Compounds 5a and 5b were further purifiedusing flash column chromatography (hexanes:ethyl acetate 4:1) to giveH-tetrazine 5a (8.9 mg, 0.023 mmol, 8.0%) and Me-tetrazine 5b (11.6 mg,0.026 mmol, 10.0%) as dark red solids.

Compound 5a: ¹H NMR (500 MHz, CD₂Cl₂) δ 10.20 (s, 1H), 8.61 (s, 1H),8.51 (dd, J=8.0, 2.0 Hz, 1H), 7.40 (d, J=8.0 Hz, 1H), 6.17 (s, 1H), 2.71(s, 3H), 2.54 (s, 3H), 2.49 (s, 3H), 2.36 (s, 3H), 2.30 (s, 3H), 2.27(s, 3H); ¹³C NMR (125 MHz, CD₂Cl₂) δ 166.5, 157.9, 154.4, 150.9, 142.3,142.1, 139.5, 138.9, 137.5, 132.5, 132.2, 131.8, 131.7, 131.2, 129.6,125.5, 121.6, 19.8, 17.2, 16.7, 15.2, 14.2, 12.8. ESIMS [M+H]+ calcd forC₂₃H₂₄BF₂N₆ 433.2, found 433.26.

Compound 5b: ¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 8.46 (dd, J=7.6,1.6 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 6.11 (s, 1H), 3.12 (s, 3H), 2.67(s, 3H), 2.56 (s, 3H), 2.46 (s, 3H), 2.38 (s, 3H), 2.26 (s, 3H), 2.23(s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 164.1, 154.5, 151.2, 141.7,141.6, 139.2, 138.4, 137.2, 132.6, 132.1, 132.1, 131.8, 131.2, 129.4,125.3, 121.7, 21.2, 20.1, 17.5, 16.7, 15.4, 14.5, 13.1. ESIMS [M−H]−calcd for C₂₄H₂₄BF₂N₆ 445.21, found 445.21.

Example 6 Characterization of TCO-Reacted 2b, 4b and 5b Characterizationof TCO-Reacted 2b

An aliquot of TCO-reacted 2b in MeCN was purified via flash columnchromatography to remove excess TCO (dichloromethane:methanol gradient20:0.1, 20:0.2, 20:0.3). ¹H NMR (400 MHz, CDCl₃) δ 7.50 (d, J=6.4 Hz,2H), 7.34 (d, J=8.0 Hz, 2H), 7.01 (s, 1H), 6.00 (s, 2H), 3.93 (m, 1H),3.02 (dd, J=10, 6.4 Hz, 1H), 2.56 (s, 6H), 2.07 (s, 3H), 2.01-1.80 (m,3H), 1.71-1.59 (m, 4H), 1.40 (s, 6H), 1.35-1.25 (m, 4H).

ESIMS [M+H]+ calcd for C₃₀H₃₆BF₂N₄O 517.30, found 517.34.

Characterization of TCO-Reacted 4b

An aliquot of TCO reacted 4b in MeCN was purified via flash columnchromatography to remove excess TCO (dichloromethane:methanol gradient20:0.1, 20:0.2, 20:0.3). ¹H NMR (400 MHz, CDCl3) δ 7.90 (d, J=8.4 Hz,1H), 7.63 (t, J=1.6 Hz, 1H), 7.23 (m, 1H), 7.22 (m, 1H), 7.05 (s, 1H),5.98 (s, 2H), 3.85 (m, 1H), 3.56 (dd, J=11.6, 3.6 Hz, 1H), 2.56 (s, 6H),2.19 (m, 2H), 1.98-1.89 (m, 2H), 1.85 (s, 3H), 1.60 (m, 3H), 1.41 (s,6H), 1.31 (m, 4H). ESIMS [M+H]+ calcd for C₃₀H₃₆BF₂N₄O 517.30, found517.32.

Characterization of TCO-Reacted 5b

An aliquot of TCO reacted 5b in MeCN was purified via flash columnchromatography to remove excess TCO (dichloromethane:methanol gradient20:0.1, 20:0.2, 20:0.3). ¹H NMR (400 MHz, CDCl3) δ 7.69 (s, 1H), 7.57(d, J=8 Hz, 1H), 7.09 (s, 1H), 7.07 (s, 1H), 6.08 (s, 1H), 3.90 (m, 1H),3.54 (dd, J=9.6, 6.8 Hz, 1H), 2.64, (s, 3H), 2.54 (s, 3H), 2.44 (s, 3H),2.33 (s, 3H), 2.18 (s, 3H), 2.13 (s, 3H), 1.94 (s, 3H), 1.86 (m, 2H),1.82-1.75 (m, 4H), 1.64-1.58 (m, 4H), 1.32 (m, 1H). ESIMS [M+H]+ calcdfor C₃₂H₃₉BF₂N₄O 545.33, found 545.38.

The quantum yield and fluorogenic activation of the compounds weremeasured and compared to the pre-TCO compounds. As shown in Table 1, thecompound exhibited a significant increase in fluorescence as compared tothe precursor compounds.

TABLE 1 Φ Φ Fluorescence Fluorescence w/TCO in w/TCO in increase inincrease in Probe water^(a) MeCN^(a) water^(b) MeCN^(b) 2b 0.80 0.23 900-fold 340-fold 4b 0.73 0.58 1600-fold 1100-fold  5b ND^(c) 0.22ND^(c) 120-fold ^(a)Quantum yield for dihydropyridazine product;fluorescein in 0.1M NaOH (pH 13, Φ = 0.925) was used as the standard.^(b)Increase in peak fluorescence intensity at reaction completion; forexperiments in water, 400 nM BODIPY-Tz, and 1 μM TCO were used.^(c)Compound 5b is insufficiently soluble in water for thisdetermination.The exceptional fluorogenic turn-on of 2 and 4—as much as 100-foldgreater than flexibly linked fluorophore-Tz conjugates—suggested thatFRET may not be the sole quenching mechanism for these compounds.

Example 7 Absorption and Fluorescence of 4b and TCO-Reacted 4b

FIG. 5 provides the absorption and fluorescence emission spectra of 4bin toluene (dielectric constant 2.4) and acetonitrile (dielectricconstant 37.5). Samples were prepared by matched dilutions from aconcentrated stock solution of 4b. For the fluorescence spectra, theexcitation wavelength was offset from peak absorbance in that solvent by10 nm; in toluene, the excitation wavelength was 496 nm; inacetonitrile, 489 nm. Emission spectra are the mean of three scans anddashed lines correspond to +/−SEM.

The activation of 4b upon reaction with TCO is demonstrated in FIG. 6,which shows the emission spectra (excitation 490 nm) of compound 4b inwater, before and after addition of TCO. For baseline and pre-TCOspectra, data plotted are means of 3 scans and dashed lines indicate+/−SEM.

Given that the fluorescence emission intensity of 4b was found to beindependent of solvent polarity, redox-based quenching, such as viaphotoinduced-electron transfer (PET) from the excited BODIPY to therelatively electron-poor tetrazine ring, was judged unlikely.

Example 8 Biological Application of Activatable BODIPY

Having advanced our understanding of the quenching mechanism, theutility of fluorogenic BODIPY-tetrazines for biological imaging wasexplored further (see FIG. 7). FIG. 7A shows fluorogenic imaging of EGFRexpression on both fixed and live A431 cells. Cells were incubated withTCO-conjugated monoclonal antibodies (see S. S. Agasti et al., Small2013, 9, 222-227), washed, and then imaged immediately after theaddition of 100 nM BODIPY-Tz in PBS. As shown in FIG. 7B, Fluorogeniclive-cell imaging of intracellular nanoparticles internalized by RAW264.7 cells. The nanoparticles are labeled with both TCO and with thenear-infrared dye VT680 (see S. S. Agasti et al., Small 2013, 9,222-227), and were imaged in two channels after addition of 100 nMBODIPY-Tz, demonstrating co-localization. Both extracellular andintracellular TCO-labeled targets were readily visualized, withexcellent signal intensity, very low background, and with no washingsteps required after addition of the dye solution.

Example 9 Preparation of Compound 7

To bromocoumarin 7a (245.0 mg, 0.733 mmol) in 8.0 mL of dioxane:water(3:1) was added 3-cyanophenylboronic acid (215.4 mg, 1.47 mmol),Pd(OAc)₂(PPh₃)₂ (27.4 mg, 0.037 mmol), and K₂CO₃ (202.6 mg, 1.46 mmol).The reaction mixture was refluxed for 7 hours after which it wasconcentrated using a rotary evaporator and purified using flash columnchromatography (hexanes:ethyl acetate gradient, 6:1 to 4:1) to give 7b(105.0 mg, 0.29 mmol, 40.1%) as a yellow solid.

¹H NMR (400 MHz, CDCl₃) δ 7.61-7.47 (m, 4H), 7.04 (s, 1H), 3.25 (m, 4H),2.89 (t, J=6.4 Hz, 2H), 2.76 (t, J=6.0 Hz, 2H), 2.16 (s, 3H), 1.97 (m,4H); ¹³C NMR (100 MHz, CDCl₃) δ 161.9, 150.5, 149.5, 146.2, 137.4,135.6, 134.5, 131.2, 129.2, 122.6, 118.9, 118.5, 118.1, 112.6, 109.0,106.7, 50.1, 49.7, 27.9, 21.7, 20.8, 20.6, 16.6. ESIMS [M+H]⁺ calcd forC₂₃H₂₁N₂O₂ 357.42, found 357.16.

Bromocoumarin 7a was prepared from literature protocol (Gong, et al.,PCT Int. Appl. (2006), WO 2006026368).

Example 10 Preparation of HyperEmissive Ligation-Initiated OrthogonalSensing (HELIOS) Probe 400 Me

To nitrile 7b (100.0 mg, 0.28 mmol) in a microwave reaction tube under astream of argon was added Zn(OTf)₂ (51.3 mg, 0.14 mmol), MeCN (0.15 mL,2.80 mmol), dioxane (0.22 mL) and NH₂NH₂ (0.44 mL, 14.0 mmol). Thevessel was sealed and allowed to stir at 60° C. for 15 hours after whichit was allowed to cool and the septum removed. To the reaction mixturewas added NaNO₂ (386.4 mg, 5.60 mmol) in 10 mL of water followed by 1 MHCl until the pH=3. The aqueous phase was extracted three times withmethylene chloride (100 mL). The combined organic extracts were driedwith MgSO₄ and concentrated using a rotary evaporator. The crude mixturewas purified using flash column chromatography (hexanes:ethyl acetategradient, 5:1 to 1:1) to give HELIOS 400Me (55.3 mg, 0.13 mmol, 46.4%)as an orange solid.

¹H NMR (400 MHz, CDCl₃) δ 8.55 (d, J=8.0 Hz, 1H), 8.50 (s, 1H), 7.63 (t,J=8.0 Hz, 1H), 7.55 (d, J=7.6 Hz, 1H), 7.06 (s, 1H), 3.26 (m, 4H), 3.07(s, 3H), 2.93 (t, J=6.4 Hz, 2H), 2.79 (t, J=6.4 Hz, 2H), 2.23 (s, 3H),1.99 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 167.4, 164.3, 162.1, 150.4,149.1, 145.6, 137.2, 135.2, 132.0, 130.3, 129.4, 127.2, 122.6, 119.9,118.6, 109.7, 107.2, 50.2, 49.8, 27.9, 21.8, 21.3, 20.9, 20.7, 16.7.ESIMS [M+H]⁺ calcd for C₂₅H₂₄N₅O₂ 426.19, found 426.24.

Example 11 Preparation of HELIOS 400H

To nitrile 7b (110.4 mg, 0.31 mmol) in a microwave reaction tube under astream of argon was added Zn(OTf)₂ (56.6 mg, 0.15 mmol), formamidineacetate (322.7 mg, 3.10 mmol), DMF (0.24 mL) and NH₂NH₂ (0.49 mL, 15.5mmol). The vessel was sealed and allowed to stir at 60° C. for 15 hoursafter which it was allowed to cool and the septum removed. To thereaction mixture was added NaNO₂ (427.8 mg, 6.20 mmol) in 10 mL of waterfollowed by 1 M HCl until the pH=3. The aqueous phase was extractedthree times with methylene chloride (100 mL). The combined organicextracts were dried with MgSO₄ and concentrated using a rotaryevaporator. The crude mixture was purified using flash columnchromatography (hexanes:ethyl acetate gradient, 4:1 to 2:1) to giveHELIOS 400H (12.1 mg, 0.029 mmol, 9.5%) as an orange solid.

¹H NMR (400 MHz, CDCl₃) δ 10.19 (s, 1H), 8.60 (d, J=7.6 Hz, 1H), 8.54(s, 1H), 7.66 (t, J=7.6 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.08 (s, 1H),3.28 (m, 4H), 2.94 (t, J=6.4 Hz, 2H), 2.81 (t, J=6.4 Hz, 2H), 2.24 (s,3H), 2.01 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 166.5, 161.9, 157.8,150.3, 148.9, 145.6, 137.2, 135.6, 131.6, 130.6, 129.4, 127.4, 122.4,119.6, 118.4, 109.4, 106.9, 50.0, 49.6, 27.8, 21.6, 20.7, 20.5, 16.5.ESIMS [M+H]⁺ calcd for C₂₄H₂₂N₅O₂ 412.17, found 412.18.

Example 7 Preparation of 7c

To bromocoumarin 7a (105.5 mg, 0.31 mmol) in 4.0 mL of dioxane:water(3:1) was added 3-cyanophenylboronic acid (91.1 mg, 0.62 mmol),Pd(OAc)₂(PPh₃)₂ (11.6 mg, 0.015 mmol), and K₂CO₃ (85.6 mg, 0.62 mmol).The reaction mixture was refluxed for 7 hours after which it wasconcentrated using a rotary evaporator and purified using flash columnchromatography (hexanes:ethyl acetate gradient, 6:1 to 4:1) to give 1c(37.7 mg, 0.10 mmol, 34.1%) as a yellow solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 7.86 (d, J=8.0 Hz, 2H), 7.48 (d, J=8.0 Hz,2H), 7.20 (s, 1H), 3.25 (m, 4H), 2.75 (m, 4H), 2.14 (s, 3H), 1.89 (m,4H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 160.2, 149.7, 149.3, 145.6, 140.8,131.8 (2C), 122.8, 118.8, 118.0, 117.3, 109.9, 108.0, 105.2, 49.2, 48.6,27.0, 20.9, 20.1, 19.9, 16.1. ESIMS [M+H]⁺ calcd for C₂₃H₂₁N₂O₂ 357.16,found 357.16.

Example 13 Preparation of HELIOS 400pMe

To nitrile 7c (37.7 mg, 0.10 mmol) in a microwave reaction tube under astream of argon was added Zn(OTf)₂ (19.3 mg, 0.52 mmol), MeCN (0.055 mL,1.05 mmol), dioxane (0.083 mL) and NH₂NH₂ (0.16 mL, 5.28 mmol). Thevessel was sealed and allowed to stir at 60° C. for 15 hours after whichit was allowed to cool and the septum removed. To the reaction mixturewas added NaNO₂ (145.9 mg, 2.11 mmol) in 10 mL of water followed by 1 MHCl until the pH=3. The aqueous phase was extracted three times withmethylene chloride (100 mL). The combined organic extracts were driedwith MgSO₄ and concentrated using a rotary evaporator. The crude mixturewas purified using flash column chromatography (hexanes:ethyl acetategradient, 4:1 to 2:1) to give HELIOS 400pMe (31.1 mg, 0.073 mmol, 73.1%)as an orange solid.

¹H NMR (400 MHz, CDCl₃) δ 8.63 (d, J=8.0 Hz, 2H), 7.51 (d, J=8.0 Hz,2H), 7.06 (s, 1H), 3.26, (m, 4H), 3.08 (s, 3H), 2.93 (t, J=6.0 Hz, 2H),2.79 (t, J=6.4 Hz, 2H), 2.34 (s, 3H), 1.99 (m, 4H); ¹³C NMR (100 MHz,CDCl₃) δ 167.4, 164.2, 161.9, 150.5, 149.0, 145.8, 140.7, 131.9 (2C),130.9, 127.9 (2C), 122.6 , 119.7, 118.5, 109.4, 106.9, 50.2, 49.8, 28.0,21.8, 21.4, 20.9, 20.7, 16.7. ESIMS [M+H]⁺ calcd for C₂₅H₂₄N₅O₂ 426.19,found 426.19.

Example 14 Preparation of 8a

To coumarin 339 (8) (410.6 mg, 1.90 mmol) dissolved in 25 mL ofacetonitrile was added NBS (373.7 mg, 2.10 mmol) and the reactionmixture allowed to stir for 2 hours. The crude mixture was concentratedusing a rotary evaporator and purified using flash column chromatography(methylene chloride to methylene chloride:methanol 250:1) to give 8a(276.4 mg, 0.94 mmol, 49.4%) as a yellow solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 7.27 (s, 1H), 6.93 (bs, 1H), 6.29 (s, 1H),3.24 (t, J=4.8 Hz, 2H), 2.72 (t, J=5.6 Hz, 2H), 2.44 (s, 3H), 1.79 (m,2H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 156.9, 152.2, 152.1, 149.4, 125.6,118.1, 107.9, 103.2, 97.0, 40.3, 26.3, 20.6, 18.9. ESIMS [M+H]⁺ calcdfor C₁₃H₁₃BrNO₂ 294.01, found 294.00. Coumarin 339 (8) was prepared fromliterature protocol (R. L. Atkins, D. E. Bliss, J. Org. Chem. 1978, 43,1975).

Example 15 Preparation of 8b

To bromocoumarin 8a (150 mg, 0.51 mmol) in 5.2 mL of dioxane:water (3:1)was added 3-cyanophenylboronic acid (151.6 mg, 1.01 mmol),Pd(OAc)₂(PPh₃)₂ (19.1 mg, 0.025 mmol), and K₂CO₃ (140.9 mg, 1.01 mmol).The reaction mixture was refluxed for 7 hours after which it wasconcentrated using a rotary evaporator and purified using flash columnchromatography (hexanes:ethyl acetate gradient, 4:1 to 2:1) to give 8b(74.5 mg, 0.23 mmol, 46.2%) as a yellow solid.

¹H NMR (400 MHz, CD₃OD) δ 7.72 (m, 1H), 7.67 (s, 1H), 7.60 (m, 2H), 7.33(s, 1H), 6.36 (s, 1H), 3.36 (t, J=5.6 Hz, 2H), 2.83 (t, J=6.4 Hz, 2H),2.23 (s, 3H), 1.93 (quin, J=5.6 Hz, 2H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ160.4, 152.9, 149.5, 149.1, 136.9, 135.7, 134.2, 131.0, 129.2, 125.7,118.7, 117.7, 116.9, 111.1, 108.2, 97.1, 40.4, 26.4, 20.7, 16.1. ESIMS[M+H]⁺ calcd for C₂₀H₁₇N₂O₂ 317.12, found 317.18.

Example 16 Preparation of HELIOS 388Me

To nitrile 8b (64.8 mg, 0.20 mmol) in a microwave reaction tube under astream of argon was added Zn(OTf)₂ (37.4 mg, 0.10 mmol), MeCN (0.11 mL,2.10 mmol), dioxane (0.16 mL) and NH₂NH₂ (0.32 mL, 10.20 mmol). Thevessel was sealed and allowed to stir at 60° C. for 15 hours after whichit was allowed to cool and the septum removed. To the reaction mixturewas added NaNO₂ (276.0 mg, 4.0 mmol) in 10 mL of water followed by 1 MHCl until the pH=3. The aqueous phase was extracted three times withmethylene chloride (100 mL). The combined organic extracts were driedwith MgSO₄ and concentrated using a rotary evaporator. The crude mixturewas purified using flash column chromatography (hexanes:ethyl acetategradient, 3:1 to 1:1) to give HELIOS 388Me (33.3 mg, 0.086 mmol, 43.2%)as an orange solid.

¹H NMR (400 MHz, CDCl₃) δ 8.56 (d, J=8.0 Hz, 1H), 8.50 (s, 1H), 7.64 (t,J=7.6 Hz, 1H), 7.56 (d, J=7.6 Hz, 1H), 7.18 (s, 1H), 6.37 (s, 1H), 4.55(s, 1H), 3.37 (m, 2H), 3.07 (s, 3H), 2.80 (t, J=6.0 Hz, 2H), 2.25 (s,3H), 1.95 (quin, J=5.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 164.3,162.1, 153.6, 149.1, 148.3, 136.9, 135.1, 132.1, 130.3, 129.5, 127.3,125.7, 120.5, 118.5, 110.6, 99.2, 41.8, 27.2, 21.7, 21.4, 16.7. ESIMS[M+H]⁺ calcd for C₂₂H₂₀N₅O₂ 386.16, found 386.13.

Example 17 Preparation of HELIOS 388H

To nitrile 8b (76.7 mg, 0.24 mmol) in a microwave reaction tube under astream of argon was added Zn(OTf)₂ (44.3 mg, 0.12 mmol), formamidineacetate (251.9 mg, 2.42 mmol), DMF (0.19 mL) and NH₂NH₂ (0.38 mL, 12.1mmol). The vessel was sealed and allowed to stir at 60° C. for 15 hoursafter which it was allowed to cool and the septum removed. To thereaction mixture was added NaNO₂ (333.9 mg, 4.84 mmol) in 10 mL of waterfollowed by 1 M HCl until the pH=3. The aqueous phase was extractedthree times with methylene chloride (100 mL). The combined organicextracts were dried with MgSO₄ and concentrated using a rotaryevaporator. The crude mixture was purified using flash columnchromatography (hexanes:ethyl acetate gradient, 3:1 to 1:1) to giveHELIOS 388H (7.48 mg, 0.020 mmol, 8.4%) as an orange solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 10.62 (s, 1H), 8.50 (d, J=8.0 Hz, 1H), 8.38(s, 1H), 7.74 (t, J=7.6 Hz, 1H), 7.62 (d, J=7.6 Hz, 1H), 7.33 (s, 1H),6.86 (s, 1H), 6.36 (s, 1H), 3.28 (m, 2H), 2.76 (t, J=6.0 Hz, 2H), 2.23(s, 3H), 1.82 (m, 2H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 165.4, 160.6,158.1, 152.9, 149.2, 149.0, 136.8, 135.1, 131.7, 129.9, 129.2, 126.7,125.7, 118.1, 117.7, 108.4, 97.2, 40.4, 26.5, 20.8, 16.2. ESIMS [M+H]⁺calcd for C₂₁H₁₈N₅O₂ 372.14, found 372.12.

Example 18 Preparation of 9b

To bromocoumarin 9a (5.25 g, 20.7 mmol) in 133.0 mL of dioxane:water(3:1) was added 3-cyanophenylboronic acid (4.56 g, 31.0 mmol),Pd(OAc)₂(PPh₃)₂ (775.3 mg, 1.03 mmol), and K₂CO₃ (5.71 g, 41.4 mmol).The reaction mixture was refluxed for 2 hours after which it wasconcentrated using a rotary evaporator. The crude was partitionedbetween water and methylene chloride and extracted 3 times (250 mL),concentrated using a rotary evaporator and purified using flash columnchromatography (methylene chloride:methanol, 10:0.05) to give 3b (2.38g, 8.6 mmol, 41.5%) as a white solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 7.82 (m, 1H), 7.78 (s, 1H), 7.64 (m, 2H),7.50 (d, J=8.4 Hz, 1H) 6.63 (d, J=8.8 Hz, 1H), 6.46 (s, 1H), 6.19 (s,2H), 2.15 (s, 3H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 160.4, 154.6, 153.1,149.6, 136.7, 135.7, 134.2, 131.1, 129.3, 126.9, 118.7, 117.6, 111.6,111.2, 108.9, 99.3, 16.2. ESIMS [M+H]⁺ calcd for C₁₇H₁₃N₂O₂ 277.09,found 277.06.

Bromocoumarin 9a was prepared from literature protocol (M. S. Schiedel,C. A. Briehn, P. Bauerle, Angew. Chem. Int. Ed. 2001, 40, 4677-4680).

Example 19 Preparation of HELIOS 327Me

To nitrile 9b (39.0 mg, 0.14 mmol) in a microwave reaction tube under astream of argon was added Zn(OTf)₂ (25.8 mg, 0.070 mmol), MeCN (0.073mL, 1.40 mmol), dioxane (0.11 mL) and NH₂NH₂ (0.22 mL, 7.00 mmol). Thevessel was sealed and allowed to stir at 60° C. for 15 hours after whichit was allowed to cool and the septum removed. To the reaction mixturewas added NaNO₂ (193.2 mg, 2.80 mmol) in 10 mL of water followed by 1 MHCl until the pH=3. The aqueous phase was extracted three times withmethylene chloride (100 mL). The combined organic extracts were driedwith MgSO₄ and concentrated using a rotary evaporator. The crude mixturewas purified using flash column chromatography (hexanes:ethyl acetategradient, 2:1 to 1:1) to give HELIOS 347Me (5.94 mg, 0.017 mmol, 12.3%)as a red solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 8.46 (d, J=7.6 Hz, 1H), 8.35 (s, 1H), 7.72(t, J=8.0 Hz, 1H), 7.60 (d, J=7.6 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 6.62(d, J=8.4 Hz, 1H), 6.48 (s, 1H), 6.16 (s, 2H), 3.00 (s, 3H), 2.22 (s,3H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 167.1, 163.2, 160.6, 154.6, 152.9,149.2, 136.5, 134.6, 131.8, 129.5, 129.2, 126.9, 126.4, 118.7, 111.5,109.0, 98.3, 20.8, 16.2. ESIMS [M+H]⁺ calcd for C₁₉H₁₆N₅O₂ 346.13, found346.11.

Example 20 Preparation of HELIOS 347H

To nitrile 9b (200.0 mg, 0.72 mmol) in a microwave reaction tube under astream of argon was added Zn(OTf)₂ (132.2 mg, 0.36 mmol), formamidineacetate (749.6 mg, 7.2 mmol), DMF (0.56 mL) and NH₂NH₂ (1.13 mL, 36.0mmol). The vessel was sealed and allowed to stir at 60° C. for 15 hoursafter which it was allowed to cool and the septum removed. To thereaction mixture was added NaNO₂ (993.6 mg, 14.4 mmol) in 15 mL of waterfollowed by 1 M HCl until the pH=3. The aqueous phase was extractedthree times with methylene chloride (150 mL). The combined organicextracts were dried with MgSO₄ and concentrated using a rotaryevaporator. The crude mixture was purified using flash columnchromatography (hexanes:ethyl acetate gradient, 2:1 to 1:1) to giveHELIOS 347H (17.6 mg, 0.053 mmol, 7.4%) as a red solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 10.61 (s, 1H), 8.50 (d, J=8.0 Hz, 1H), 8.39(s, 1H), 7.75 (t, J=8.0 Hz, 1H), 7.63 (d, J=7.6 Hz, 1H), 7.52 (d, J=8.4Hz, 1H), 6.62 (dd, J=8.8, 2.4 Hz, 1H), 6.48 d (J=2.0 Hz, 1H), 6.16 (s,2H), 2.23 (s, 3H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 165.4, 160.6, 158.1,154.6, 152.9, 149.2, 136.6, 135.0, 131.8, 129.8, 129.2, 126.9, 126.7,118.6, 111.5, 109.0, 98.3, 16.2. ESIMS [M+H]⁺ calcd for C₁₈H₁₄N₅O₂332.11, found 332.09.

Example 21 Preparation of 10a

To difluorinated hydroxycoumarin (Marina Blue®) (10) (1.06 g, 4.99 mmol)dissolved in 50 mL of acetonitrile was added NBS (0.93 g, 5.24 mmol) andthe reaction mixture allowed to stir for 1 hour. The crude mixture wasconcentrated using a rotary evaporator and purified using flash columnchromatography (hexanes:ethyl acetate, 2:1) to give 10a (1.32 g, 4.52mmol, 90.6%) as a white solid.

¹H NMR (400 MHz, MeOD) δ 7.30 (d, J=11.2 Hz, 1H), 2.51 (s, 3H); ¹³C NMR(100 MHz, MeOD) δ 147.9, 143.3 (t, J=2.8 Hz), 141.0 (dd, J=234.7, 5.0Hz), 131.3 (dd, J=216.0, 6.4 Hz), 129.9 (m, 2C), 102.7 (d, J=9.1 Hz),101.7, 97.7 (dd, J=19.0, 3.3), 10.4. ESIMS [M−H]⁻ calcd for C₁₀H₄BrF₂O₃288.93, found 288.88.

Example 21 Preparation of 10b

To bromocoumarin 10a (475.9 mg, 1.63 mmol) in 15.0 mL of dioxane:water(3:1) was added 3-cyanophenylboronic acid (479.0 mg, 3.26 mmol),Pd(OAc)₂(PPh₃)₂ (61.2 mg, 0.082 mmol), and K₂CO₃ (225.3 mg, 3.26 mmol).The reaction mixture was refluxed for 7 hours after which it wasconcentrated using a rotary evaporator and purified using flash columnchromatography (hexanes:ethyl acetate gradient, 2:1 to 1:1) to give 4b(142.9 mg, 0.45 mmol, 28.0%) as a white solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 7.89 (t, J=3.6 hz, 1H), 7.83 (s, 1H), 7.69(m, 2H), 7.60 (d, J=11.6 Hz, 1H), 2.21 (s, 3H); ¹³C NMR (100 MHz,(CD₃)₂SO) δ 158.7, 148.8 (t, J=2.6 Hz), 148.6 (dd, J=232.6, 5.2 Hz),139.1 (dd, J=235.8, 6.8 Hz), 138.5 (dd, J=7.6, 1.8 Hz), 137.6 (dd,J=12.7, 5.3 Hz), 135.7, 135.3, 133.8, 131.8, 129.5, 122.6, 118.5, 111.4,111.1 (d, J=8.9 Hz), 107.0 (dd, J=18.6, 2.9 Hz), 16.6. ESIMS [M+H]⁺calcd for C₁₇H₁₀F₂NO₃ 314.06, found 314.01.

Example 22 Preparation of 4c

To nitrile 10b (91.1 mg, 0.29 mmol) in 3.0 mL of methylene chloride wasadded N,N-Diisopropylethylamine (0.15 mL, 0.87 mmol) and DMAP (1.77 mg,0.014 mmol). The mixture was then cooled to 0° C. and bromomethyl methylether (0.059 mL, 0.72 mmol) was added dropwise. The ice bath was removedand the reaction was allowed to stir at room temperature for one hourafter which it was concentrated using a rotary evaporator and purifiedusing flash column chromatography (hexanes:ethyl acetate 4:1) to give10c (99.9 mg, 0.28 mmol, 96.4%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J=7.6 Hz, 1H), 7.58 (s, 1H), 7.54 (m,2H), 7.20 (dd, J=8.8, 2.0 Hz, 1H), 5.28 (s, 2H), 3.59 (s, 3H), 2.25 (s,3H); ¹³C NMR (100 MHz, CDCl₃) δ 159.0, 152.5 (dd, J=241.0, 4.0 Hz),147.8 (t, J=2.6 Hz), 144.1 (dd, J=246.7, 5.8 Hz), 139.1 (dd, J=8.1, 2.4Hz), 136.4 (dd, J=11.2, 4.7 Hz), 135.4, 134.8, 133.9, 132.3, 129.7,125.7, 118.5, 116.1 (d, J=8.8 Hz), 113.2, 106.5 (dd, J=18.6, 3.8 Hz),99.3 (t, J=3.8 Hz), 57.6, 17.1. ESIMS [M+H]⁺ calcd for C₁₉H₁₄F₂NO₄358.08, found 358.04.

Example 23 Preparation of HELIOS 370Me

To nitrile 10c (144.0 mg, 0.40 mmol) in a microwave reaction tube undera stream of argon was added Zn(OTf)₂ (73.6 mg, 0.201 mmol), MeCN (0.21mL, 4.03 mmol), dioxane (0.32 mL) and NH₂NH₂ (0.63 mL, 20.1 mmol). Thevessel was sealed and allowed to stir at 60° C. for 15 hours after whichit was allowed to cool and the septum removed. To the reaction mixturewas added NaNO₂ (556.1 mg, 8.06 mmol) in 10 mL of water followed by 1 MHCl until the pH=3. The aqueous phase was extracted three times withmethylene chloride (100 mL). The combined organic extracts were driedwith MgSO₄ and concentrated using a rotary evaporator. The crude mixturewas filtered through 10 g of silica (methylene chloride:methanol,100:0.5, 100 mL) and concentrated using a rotary evaporator. Thismixture was then dissolved in 8 mL of methylene chloride and TFA (1 mL)was added and the reaction was allowed to stir at room temperature for30 minutes, after which it was concentrated under a stream of nitrogen.The crude mixture was purified using flash column chromatography(methylene chloride:methanol, 10:0.1) to give HELIOS 370Me (77.4 mg,0.20 mmol, 50.6%) as a red solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 8.50 (d, J=7.60 Hz, 1H), 8.40 (s, 1H), 7.76(t, J=7.6 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.55 (m, 1H), 3.01 (s, 3H),2.26 (s, 3H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 167.2, 163.1, 158.9, 148.6(dd, J=233.0, 5.3 Hz), 148.3 (t, J=2.6 Hz), 139.1 (dd, J=233.1, 9.3 Hz),138.5 (dd, J=7.5, 2.0 Hz), 137.4 (m), 135.5, 134.2, 131.9, 129.4, 129.1,126.9, 123.6, 111.3 (d, J=9.3 Hz), 107.0 (dd, J=19.0, 2.7 Hz), 20.8,16.7. ESIMS [M−H]⁻ calcd for C₁₉H₁₁F₂N₄O₃ 381.08, found 381.06.

Example 24 Preparation of HELIOS 370H

To nitrile 10c (144.0 mg, 0.40 mmol) in a microwave reaction tube undera stream of argon was added Zn(OTf)₂ (73.6 mg, 0.201 mmol), formamidineacetate (419.6 mg mL, 4.03 mmol), DMF (0.32 mL) and NH₂NH₂ (0.63 mL,20.1 mmol). The vessel was sealed and allowed to stir at 60° C. for 15hours after which it was allowed to cool and the septum removed. To thereaction mixture was added NaNO₂ (556.1 mg, 8.06 mmol) in 10 mL of waterfollowed by 1 M HCl until the pH=3. The aqueous phase was extractedthree times with methylene chloride (100 mL). The combined organicextracts were dried with MgSO₄ and concentrated using a rotaryevaporator. The crude mixture was filtered through 10 g of silica(methylene chloride:methanol, 100:0.5, 100 mL) and concentrated using arotary evaporator. This mixture was then dissolved in 8 mL of methylenechloride and TFA (1 mL) was added and the reaction was allowed to stirat room temperature for 30 minutes, after which it was concentratedunder a stream of nitrogen. The crude mixture was purified using flashcolumn chromatography (methylene chloride:methanol, 10:0.1) to giveHELIOS 370H (26.9 mg, 0.073 mmol, 18.3%) as a red solid.

¹H NMR (400 MHz, (CD₃)₂SO) δ 10.61 (s, 1H), 8.53 (d, J=8.0 Hz, 1H), 8.44(s, 1H), 7.78 (t, J=7.6 Hz, 1H), 7.67 (d, J=7.6 Hz, 1H), 7.60 (d, J=11.6Hz, 1H), 2.27 (s, 3H); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 165.3, 158.9,158.2, 148.5 (dd, J=232.0, 5.1 Hz), 148.5 (t, J=2.4 Hz), 139.1 (dd,J=235.6, 7.2 Hz), 138.5 (dd, J=9.4), 137.4 (m), 135.5, 134.6, 131.9,129.5 (2C), 127.3, 123.6, 111.3 (d, J=8.9 Hz), 107.0 (dd, J=21.8, 2.8Hz), 16.7. ESIMS [M−H]⁻ calcd for C₁₈H₉F₂N₄O₃ 367.06, found 366.96.

Example 25 Preparation of TCOc

To TCO-NHS (45.4 mg, 0.17 mmol) dissolved in 2 mL of methylene chloridewas added triethylamine (0.05 mL, 0.35 mmol), and2-(2-Aminoethoxy)ethanol (0.07 mL, 0.71 mmol). The mixture was allowedto stir at room temperature for thirty minutes after which it wasconcentrated under a stream of nitrogen. The crude mixture was purifiedusing flash column chromatography (hexanes:ethyl acetate gradient, 1:1to 100% ethyl acetate) to give TCOc (43.3 mg, 0.073 mmol, 98.8%) as aclear oil.

¹H NMR (400 MHz, CDCl₃) δ 5.50 (m, 2H), 5.03 (m, 1H), 4.31 (dd, J=9.6,6.0 Hz, 1H), 3.71 (m, 2H), 3.53 (m, 4H), 3.33 (m, 2H), 2.31 (m, 4H),2.01-1.85 (m, 4H), 1.77-166 (m, 2H), 1.55-1.51 (m, 1H); ¹³C NMR (100MHz, CDCl₃) δ 156.6, 135.1, 133.2, 80.9, 72.4, 70.4, 61.9, 41.3, 40.9,38.8, 34.5, 32.7, 31.1. ESIMS [M+H]⁺ calcd for C₁₃H₂₄NO₄ 258.17, found258.37.

Example 26 Preparation of Phalloidin-TCO

To a 10 mM solution of TCO-PEG4-NHS (70 μL, 0.7 μmoles, Click ChemistryTools, Scottsdale, Ariz.) in DMF in a microvial was addedamino-phalloidin (60 μg, 0.07 μmoles, American Peptide Company,Sunnyvale, Calif.) and diisopropylethylamine (DIPEA, 0.2 μL, 1.1 μmole).After 30 minutes at room temperature with occasional vortex agitation,the reaction mixture was purified by reverse phase chromatography on aWaters Xterra C18, 2.5 μm, 10 mm×50 mm, column (water:acetonitrile, bothwith 0.1% formic acid; gradient elution from 5% to 75% acetonitrile) togive phalloidin TCO (58 μg, 0.05 μmoles, 70%). The amounts ofaminophalloidin and phalloidin-TCO product were determinedspectrophotometrically, based on the known extinction coefficient ofphalloidin at 291 nm (13,500). Reverse phase LCMS characterization ofthe purified material: ESI-MS [M+H]⁺ calculated for C₅₅H₈₂N₁₀O₁₇S1187.56, found 1187.43.

Example 27 Preparation of Marina Blue-Tz

To a solution of Marina Blue-succcinimidyl ester (LifeTechnologies,M10165, Grand Island, N.Y.) at 10 mM in DMF (50 μL, 0.5 μmoles) wasadded 1 uL of diisopropylethylamine, followed by a small aliquot of drybenzylaminotetrazine-HCl (MW 223.06). After 30 minutes at roomtemperature with occasional vortex agitation, the reaction mixture waspurified by reverse phase chromatography on a Waters Xterra C18, 2.5 μm,10 mm×50 mm, column (water:acetonitrile, both with 0.1% formic acid;gradient elution from 5% to 75% acetonitrile) to give Marina Blue-Tz(yield not determined). Reverse phase LCMS characterization of thepurified material: ESI-MS [M−H]⁻ calculated for C₂₁H₁₅F₂N₅O₄ 438.11,found 438.01.

Example 28 Fluorogenic Characterization of HELIOS Probes

As described above, the fluorescence purity of all HELIOS compounds wasverified by LCMS prior to quantitative activation experiments, for whicha fresh aliquot of the fluorophore collected from the analytical HPLCelution was used. Exceptionally pure material is required to obtain thepeak measured turn-on ratios, as the presence of trace brightcontaminants limits the maximum observable ratio.

Stock solutions of the freshly-purified tetrazine dyes were prepared inMeCN and stored in the dark at 4° C. during experiments. Forfluorescence measurements, the probes diluted into 2 mL or 3 mL ofphosphate buffered saline (PBS), pH 7.4 (Corning, cellgro) in a standard10 mm quartz cuvette. Working at peak excitation and emissionwavelengths for each probe, data were collected as a continuous timeseries to enable accurate measurement of the baseline intensity valuesand optimize signal to noise. Fluorescence experiments were conducted ata range of dye concentrations spanning 100 nM-750 nM, with 500 nM beinga typical working concentration. The time to peak turn on ratio (but notthe final magnitude) is a function of the added TCOc (see Example 25)concentration; for the time courses presented herein, 10 μM TCOc wasused. Measurements of solvent and pre-activation emission intensity forbaseline values were collected serially over at least 30 seconds, priorto addition of TCOc to initiate the fluorogenic reaction. After additionof TCOc (typically a 20-fold excess, as above), the fluorescenceemission intensity was monitored until a plateau was reached. Activationratios were calculated from the peak emission intensity of thedihydropyridazine product and the corresponding baseline intensity overbackground. Data were normalized to set the initial backgroundfluorescence of the HELIOS probe to one unit over background, as plottedin FIG. 9 in the main text.

Quantum yield determinations: quinine sulfate dihydrate (FluorescenceReference Standard grade, AnaSpec, Inc) in 0.5 M H₂SO₄ was used as areference, with an excitation wavelength of 370 nm; a value of 0.546 wasused for the reference quantum yield (Eaton, D. F., Pure and AppliedChemistry, 1988 60(7), 1107-1114). Calculations were made according tothe methods described by Crosby and Demas (Chemical Reviews, 1971,75(8), 991-1024).

HELIOS 400Me and HELIOS 400pMe were both soluble in phosphate bufferedsaline (PBS) at micromolar concentrations and negligibly fluorescent intheir native state. After rigorous purification to remove tracefluorescent impurities, the fluorogenic properties of these newcoumarin-Tz conjugates was evaluated on reaction with TCOc, a noveltrans-cyclooctene derivative (TCOc) that incorporates a carbamate-linkedPEG₂ side chain for improved water solubility. Addition of TCOc toHELIOS 400Me in PBS yielded a 4,000-fold peak turn-on ratio (FIG. 9).Reaction of HELIOS 400pMe with TCOc yielded a turn on ratio of1,000-fold, four-fold lower than its meta-linked counterpart.Mechanistically, the observation of 1000-fold turn-on in aperpendicular-dipole configuration argues against FRET playing asignificant role in the energy transfer mechanism. As shown below,HELIOS 388Me in PBS yielded a remarkable 11,000-fold turn-on uponreaction with TCOc, the highest turn-on ratio reported to date. Whenligated to TCOc in PBS, HELIOS 347Me and HELIOS 370Me displayed2,500-fold and 2,900-fold turn-on ratios, respectively, in spite ofminimal spectral overlap with the tetrazine absorption band at 520 nm.By comparison, FRET-based coumarin-tetrazine interactions aredramatically less efficient: a flexibly linked analogue of HELIOS 370Hdisplayed only a 60-fold turn-on (Marina Blue-Tz), corroborating thedipole-orientation analysis described previously. All four HELIOS probesexhibit very good postclick quantum yields in PBS, in agreement withstructurally similar coumarins (Table 2).

TABLE 2 Peak Ex/Em Φ Fluorescence Probe Wavelengths ε^(a) w/TCOc^(b)Enhancement^(c) HELIOS 400 400/502 16,000 0.41 4,000-fold HELIOS 388388/482 20,000 0.38 11,000-fold  HELIOS 370 370/463 19,000 0.492,900-fold HELIOS 347 347/455 18,500 0.29 2,500-fold Marina Blue-Tz362/459 ND ND  60-fold ^(a)At peak excitation wavelength in PBS, pH 7.4.^(b)Quantum yield for the dihydropyridazine product after completereaction of the indicated compound with TCOc in PBS at pH 7.4; quininesulfate in H₂SO₄ (0.5M, Φ = 0.546) was used as the standard.^(c)Fluorogenic turn-on ratio of the Me-tetrazines upon reaction withTCOc.

Example 29 Solvent Polarity Effects

Fluorescence emission spectra of HELIOS 400Me in PBS (pH 7.4),acetonitrile (dielectric constant 37.5), and toluene (dielectricconstant 2.4) was measured (see FIG. 10). Emission spectra are the meanof 2 or 3 scans and the dashed lines represent +/−SEM. Instrumentsettings were adjusted to optimize sensitivity given the minimalfluorescence of the native HELIOS probe, and samples were prepared bymatched dilution of a concentrated stock solution of HELIOS 400Me intothe respective solvents. Redox-based quenching, such as throughphotoinduced electron transfer (PET) from the excited coumarin to therelatively electron-poor tetrazine ring, was judged unlikely tocontribute significantly, because the fluorescence emission intensitywas largely independent of solvent polarity, with less than a twofoldchange between PBS and the organic solvents, and no intensity differencebetween toluene (ε=2.4) and acetonitrile (ε=37.5). PET ischaracteristically enhanced by the relative stabilization ofchargeseparated states in polar solvents [E. E. Neuteboom, S. C. J.Meskers, E. H. A. Beckers, S. Chopin, R. A. J. Janssen, J. Phys. Chem. A2006, 110, 12363].

Example 30 In Vitro Imaging General Methods

-   Microscope: Multichannel images were collected on an Olympus    Fluoview FV1000 confocal laser microscope. Coumarin probes were    excited with a 405 nm laser, with alternate excitation sources used    as relevant for reference channels, paired with appropriate emission    filter sets.-   Cell culture: A-431 cells (ATCC CRL-1555) and COS-1 cells (ATCC    CRL-1650) were cultivated in Dulbecco's Modified Eagle's Medium    (DMEM), supplemented with 10% fetal bovine serum and grown in    standard culture conditions in 10 cm dishes. OVCA-429 cells were    cultivated in RPMI-1640 supplemented with 10% fetal bovine serum    under standard culture conditions. For imaging experiments cells    were plated on Millicell EZ slides (EMD Millipore, Inc, Billerica,    Mass.).-   Antibody reagents: Monoclonal antibody-TCO conjugates were prepared    by incubation of commercially available monoclonal antibodies    TCO-PEG4-NHS (Click Chemistry Tools, Scottsdale, Ariz.). Anti EGFR    (Cetuximab, Imclone). Anti cytochrome c oxidase (COXIV, Cell    signaling Technology, #4844, Danvers, Mass.).-   An aliquot of antibody in the manufacturer-supplied storage solution    was buffer exchanged into PBS with 10 mM sodium bicarbonate, pH 8.0,    on a 40K ZebaSpin desalting column (0.5 mL, Thermo Fisher    Scientific, Rockford, Ill.). To this solution was added 20    equivalents of TCO-PEG4-NHS; the mixture was allowed to react at    25° C. for 30 minutes, with continuous shaking. The reaction mix was    loaded onto a 40K ZebaSpin column to remove organic solvent and    small molecule fractions; this eluate was loaded onto a second 40K    ZebaSpin column to ensure comprehensive removal of any excess TCO.-   EGFR Imaging: Fixed A431 cells were prepared by treatment with 4%    paraformaldehyde solution (10 min, room temperature), followed by 3    washes with PBS. Fixed cells were stored at 4° C. until the time of    imaging, when they were incubated for 20 minutes with 20 μg/mL    cetuximab-TCO, then rinsed three times with PBS.-   For optimal image quality, HELIOS 370H probe must be purified on the    day of imaging by reversed phase HPLC-MS. The concentration of stock    solutions in PBS were calculated by absorbance spectrometry, based    on the measured extinction coefficient of 19000 M⁻¹ cm⁻¹. Prior to    imaging, the purified stock solutions were subjected to turn-on    testing, verifying a fluorogenic turn-on ratio of >1000-fold for    HELIOS 370H. For imaging experiments, the acetonitrile stocks were    diluted into PBS to yield a 100 nM solution. Image acquisition:    Immediately prior to imaging, buffer was replaced with a 100 nM    solution of HELIOS 370H probe in PBS. Specific staining was evident    within 10 seconds and reached maximum signal/background intensity    over a time course of 3-5 minutes.-   Mitochondria Imaging: At ˜70% confluence, OVCA-429 cells were    incubated with 3% v/v of CellLight Fluorescent mitochondria-targeted    red fluorescent protein BacMam reagent, reconstituted according to    the manufacturer's guidelines (C10601, Invitrogen, Carsbad, Calif.),    in complete medium for 24 h. Following incubation, cells were washed    in PBS and incubated in growth media a further 24 hrs before    fixation.-   The cells were fixed with 4% paraformaldehyde and permeabilized with    0.5% Triton-X-100 in PBS. Cells were incubated with anti-COX IV-TCO    (10 μg/mL) for 40 minutes, then washed twice with PBS prior to    imaging. HELIOS 388H was freshly purified as described above and    added to cells at 100 nM concentration. Mitochondrial labeling was    evident within 2 minutes and stable target to background ratios were    observed on serial images collected up to an hour from dye addition.-   Actin Cytoskeleton Imaging: carried out per procedures developed by    Mitchison and coworkers (e.g. Cramer, L., and Mitchison, T. J., Cell    Biol. 1993 August; 122(4):833-43,    http://mitchison.med.harvard.edu/protocols.html). In brief,    phalloidin-TCO was dissolved in methanol to prepare a stock solution    at 250-1000 μg/mL (stored at −80° C.); this stock was diluted into    the labeling buffer to give a final staining solution at 1 μg/mL.    Labeling buffer: 10 mM Tris buffered saline, pH 7.4 (TBS), with 0.1%    triton X-100 and 2% bovine serum albumin.-   COS-1 cells were grown in standard culture conditions as described    above and then fixed and permeabilized per the procedures of    Mitchison and coworkers (vide supra). In brief, cells were fixed in    4% formaldehyde in cytoskeleton buffer for 20 minutes, and then    permeabilized with 0.1% Triton-X-100 in TBS. Cytoskeleton buffer: 10    mM MES, pH6.1, 138 mM KCl, 3 mM MgCl, 2 mM EGTA, 0.32M sucrose.    Nuclear staining was performed by incubating the fixed and    permeabilized cells with DRAQS (Biostatus, DR50050) diluted to a    final concentration of 1 μM for 3-5 minutes at room temperature.    After 20-40 minutes incubation with phalloidin-TCO (1 μg/mL), cells    were washed once with PBS and then imaged after addition of 100 nM    HELIOS 388H or HELIOS 370H.

Results

HELIOS 370H was used as a model to determine the applicability of theHELIOS probes as a native bioorthogonal fluorogenic imaging agent. Thismodel system was used to assess HELIOS probe kinetics in theextracellular context: imaging of the epidermal growth factor receptor(EGFR) on the surface of cancer cells. EGFR overexpression plays acritical role in the most common molecularly-defined subtype of lungcancer, where it is a key treatment target, and drives proliferation inother epithelial malignancies, including colon cancer and pancreaticcancer. As noted above, A431 cells were incubated with a TCO labeledanti-EGFR antibody (20 μg/mL, Cetuximab, ImClone) for 20 minutes andthen washed briefly with PBS. Addition of 100 nM HELIOS 370H in PBSrevealed bright, membrane specific staining coinciding with the knowndistribution of the receptor (FIG. 11A). Images were generated withinseconds of dye addition and exhibited no nonspecific binding even afterextended incubation, nor any membrane staining in the presence of acontrol antibody-TCO conjugate (FIG. 11B).

To further explore the imaging potential of HELIOS probes, mitochondriawere selected as a target; their structures have features at thediffraction limit of conventional light microscopy. OVCA-429 cellsexpressing mitochondria-specific red fluorescent protein (RFP) wereincubated with an anti-mitochondria antibody-TCO conjugate andvisualized with HELIOS 388H, yielding high spatial resolution imageswith good colocalization (FIG. 12A). Intracellular imaging of smallmolecule targets is another area of intense interest, given thepotential applications in drug development, chemical biology, andoptical pharmacology. To demonstrate the utility of HELIOS probes inthis context with a structurally validated model system, the ability ofthe probes to image the actin cytoskeleton was tested with aphalloidin-TCO conjugate (Example 26). Sequential addition ofphalloidin-TCO and several HELIOS probes produced vivid fluorogenicimages of the cytoskeleton; control experiments revealed negligiblebackground (FIG. 12B).

Example 31 In Vivo Imaging with HELIOS Probes

Nude mice (Cox7, Massachusetts General Hospital) were surgicallyimplanted with a dorsal skin window chamber. A-431 cells (ATCC CRL-1555,Manassas, Va.), a human epidermoid carcinoma cell line withoverexpression of the epidermal growth factor receptor (EGFR), were thenimplanted into the window chamber as a suspension of 2-3 million cellsin a 1:1 mixture of phosphate buffered saline (PBS) and matrigel. Tumorswere allowed to develop for ˜2 weeks, by which time they had becomevascularized and attained a diameter of 2-3 mm.

In parallel, a TCO conjugated anti-EGFR antibody was prepared aspreviously reported (Haun et al., Nat. Nanotech., 2010, 5, 660-665). 24hours prior to imaging mice were injected via tail vein IV catheter with100 μL of a 1 mg/mL solution of the antibody in PBS. On the day ofimaging, a derivative of compound 2a:

was purified by HPLC. The pure dye was dried by evaporation andformulated by dissolving the dry powder in 20 μL of a 1:1 mixture ofdimethylacetamide:solutol HS-15, followed by slow dilution in PBS to afinal volume of 300 μL. The concentration of the clear orange solutionwas determined spectrophotometrically (between 100-200 μM) and stored at4° C. until imaging.

Mice were anesthetized with 2% isoflurane in 2 L/min oxygen on a heatedmicroscope stage and a tail vein catheter was placed. In some instances,Angiosense-680 (Perkin Elmer, Waltham, Mass., USA) was injected todelineate the vasculature. Static and time series images were collectedusing a customized Olympus FV1000 confocal microscope (Olympus America).A XLUMPLFLN 20× water immersion objective (NA 1.0) water immersionobjective was used for data collection (Olympus America). Pre-treatmentimages of the tumor cells were collected, and then time-lapse imagingwas initiated synchronously with injection of 150 μL of the fluorogenicBODIPY-tetrazine dye by tail vein IV, followed by a second 150 μLinjection of dye ten minutes later. Images in FIG. 13 illustrate brightmembrane-specific staining of tumor cells within the window chamber,consistent with the known distribution of EGFR, as well as punctate fociwithin the cells at site of endocytic receptor internalization, all withexcellent target to background ratios.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A compound of Formula (I):F-L-Tz or a salt thereof, wherein: F is a fluorophore selected from thegroup consisting of:

wherein: R¹ and R² are selected from the group consisting of: H,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₁-C₆)alkoxy, —COR¹⁰, —CO₂R¹⁰, —SOR¹², —SO₂R¹², —NR¹⁰R¹¹, —NO₂,(C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 membered heterocyclyl, and 5-10membered heteroaryl, each of which is independently substituted orunsubstituted, and wherein if both R¹ and R² are present, no more thanone of R¹ and R² is H; R³, R⁴, R⁵, R⁶, and R⁷ are independently selectedfrom H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₁-C₆)alkoxy, -SO3H, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 memberedheterocyclyl, 5-10 membered heteroaryl, each of which is independentlysubstituted or unsubstituted, and a reactive moiety; R⁸ and R⁹ areindependently selected from halogen, (C₁-C₆)alkyl, (C₂-C₆)alkynyl,—CO₂R¹⁰, (C₁-C₆)alkoxy, O(4 membered heterocyclyl),—O—(C₁-C₆)alkyl-O(nPEG), each of which is independently substituted orunsubstituted; PEG is a polyethylene glycol polymer; each R¹⁰ and R¹¹are independently selected from H and (C₁-C₆)alkyl; and each R¹² isindependently a (C₆-C₁₀)aryl; L is a conjugated linker; and Tz is asubstituted or unsubstituted tetrazine; wherein the linker bridges theTz and F moieties in a single conjugated pi-system.
 2. The compound ofclaim 1, or a salt thereof, wherein the single conjugated pi-system isnon-coplanar.
 3. The compound of claim 2, or a salt thereof, whereinsteric factors enforce a twist in the L-Tz bond to F and originate fromsubstituents on either the linker or on the fluorophore.
 4. The compoundof claim 1, or a salt thereof, wherein the L-Tz moiety is oriented withrespect to the F moiety such that the tetrazine transition dipole iseither collinear with or parallel to the transition dipole of thefluorophore. 5.-6. (canceled)
 7. The method of claim 1, or a saltthereof, wherein R¹ and R² are independently selected from methyl,ethyl, isopropyl, and tert-butyl.
 8. The method of claim 1, or a saltthereof, wherein R³, R⁴, R⁵, R⁶, and R⁷ are independently selected fromH, methyl, ethyl,

9.-26. (canceled)
 27. The compound of claim 1, or a salt thereof,wherein the linker is an aromatic linker.
 28. The compound of claim 27,or a salt thereof, wherein the aromatic linker is selected from thegroup consisting of:

wherein: R^(1a), R^(2a), R^(3a), and R^(4a) are selected from the groupconsisting of: H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₁-C₆)alkoxy, —COR¹³, —CO₂R¹³, —SOR¹⁵, —SO₂R¹⁵,—NR¹³R¹⁴, —NO₂, (C₃-C₁₀)carbocyclyl, (C₆-C₁₀)aryl, 4-10 memberedheterocyclyl, and 5-10 membered heteroaryl, each of which isindependently substituted or unsubstituted.
 29. The compound of claim 1,or a salt thereof, wherein Tz is a moiety having the structure:

wherein: R^(1b) is selected from the group consisting of: H,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₆-C₁₀)aryl, and 5-10 memberedheteroaryl, each of which is independently substituted or unsubstituted.30. The compound of claim 29, or a salt thereof, wherein the Tz moietyis selected from the group consisting of:


31. The compound of claim 1, wherein the compound of Formula (I) isselected from the group consisting of:

or a salt thereof. 32.-35. (canceled)
 36. A method of imaging a subject,the method comprising: a) administering to the subject an effectiveamount of a dienophile conjugated to one or more of a small moleculetherapeutic agent, antibody, nanoparticle, polymer, and mixturesthereof; b) administering to the subject an effective amount of acompound of claim 1, or a salt thereof; and c) imaging the subject. 37.The compound of claim 1, or a salt thereof, wherein F is:


38. The compound of claim 37, or a salt thereof, wherein R¹ and R² areeach methyl.
 39. The compound of claim 37, or a salt thereof, whereinR³, R⁴, R⁵, R⁶, and R⁷ are independently selected from H, methyl, andethyl.
 40. The compound of claim 37, or a salt thereof, wherein L is:

wherein R^(1a), R^(2a), R^(3a), and R^(4a) are each independentlyselected from the group consisting of H and (C₁-C₆)alkyl.
 41. Thecompound of claim 37, or a salt thereof, wherein Tz is a moiety havingthe structure:

wherein R^(1b) is selected from the group consisting of H and(C₁-C₆)alkyl.
 42. A compound, which is:

or a salt thereof.
 43. A pharmaceutical composition comprising thecompound of claim 42, or a salt thereof, and a pharmaceuticallyacceptable excipient.
 44. A method of imaging a subject, the methodcomprising: a) administering to the subject an effective amount of adienophile conjugated to one or more of a small molecule therapeuticagent, antibody, nanoparticle, polymer, and mixtures thereof; b)administering to the subject an effective amount of the compound ofclaim 42, or a salt thereof; and c) imaging the subject.